Blood biomarkers for respiratory infections

ABSTRACT

Methods and kits for diagnosing and/or treating a lower respiratory infection in a subject include obtaining a biological sample from the subject; detecting RNA expression levels of one or more biomarkers in the biological sample and comparing the expression levels of the one or more three biomarkers to at least one invariant control marker wherein an increase or decrease in the level of expression of the one or more biomarkers as compared to the at least one invariant control marker is indicative of a lower respiratory infection.

CROSS-REFERENCE OF RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/542,863, filed Jul. 11, 2017, now allowed, which is a U.S. NationalPhase Application under 35 U.S.C. § 371 of PCT/US2016/012981, filed Jan.12, 2016, the entire content of which is hereby incorporated byreference and this application claims priority to U.S. ProvisionalApplication No. 62/102,350 filed Jan. 12, 2015, the entire contents ofwhich are hereby incorporated by reference.

BACKGROUND Technical Field

The field of the currently claimed embodiments of this invention relateto methods and kits for assessing and treating lower respiratoryinfections in a subject, and more particularly to assessing and treatinglower respiratory infections in a subject using the analysis ofbiomarkers isolated from the subject.

Discussion of Related Art

Worldwide, communicable diseases account for 10 million deaths per year[1J. An important step in controlling this problem is developing simple,inexpensive, and rapid diagnostics. Attempts to develop assays based oncirculating biomarkers for infection or inflammation in plasma have beenlargely unsuccessful A major weakness in tills approach is that the keybiomarkers are retained within the activated immune cells, particularlyprimed or activated neutrophils.

Of the 10 million deaths from communicable diseases, the largestcategory are lower respiratory infections (LRI), such as influenza, andpneumonia, accounting, for 2.8 M deaths per year [1]. By comparison,H1V-related diseases are a close second, with 2.6 M, but diarrhealdiseases cause half as many deaths (1.4 M). Surprisingly, fever andelevated white blood cell count (WBC) are very poor metrics ofpneumonia. Fever, for in stance, does not occur when an infection ishighly localized from the immune system, as occurs in encapsulated lunginfections, such as TB. Likewise, other bacteria can form ‘hiofilms’ inwhich they fuse together (i.e. fusobacteria), and secrete viscousprotective coatings that can hinder immune cell response and antibioticpenetration. Further, fever is easily masked by anti-inflammatorymedications such as aspirin. Importantly, even if present, fever andelevated WBC do not detect whether the person has a viral versusbacterial infection, a critical factor in the decision whether b useantibiotics or not. In developing countries, and even in rural areas ofdeveloped countries, access to imaging equipment, such as X-ray orcomputed tomography (CT), can be limited. Often, a stethoscope is theonly available means to diagnose pulmonary dysfunction, but it haslimited ability to distinguish infections from allergy, asthma, orbronchitis and depends primarily on the clinical skills and knowledge ofthe healthcare personnel,

There is a medical need for a high sensitivity test for infections.There is a growing list of diagnostics that detect specific infections,such as streptococci, sometimes at the point of care. Surprisinglyhowever, there are no commercial blood tests that accurately detectinfections in a general way, which could then be used to justify testsfor specific pathogens. Recent studies, have described a method forusing next-generation sequencing (NGS) to specifically identify thepathogens within a sample of sputum from intubated ICU patients [2],However, it costs >$300 per test arid takes days, without actuallyknowing whether the patient has a bacterial infection versus bronchialinflammation. Thus, there is a great need for a highly sensitive, butpathogen-agnostic test for internal infections of, for example, thelungs, appendix, central nervous system (CNS), kidneys, and otherorgans. Once a test is positive, then it is worthwhile to identify thepathogen, whether viral or bacterial, and determine its antibioticsensitivity.

SUMMARY

Embodiments of the present invention include a method of diagnosing alower respiratory infection in a subject, including the steps; obtaininga biological sample from the subject; detecting expression levels of oneor more biomarkers in the biological sample selected from the groupconsisting of Neutrophil defensin 1 precursor, Defensin alpha 1,LOC6536G0, defensin alpha IB, Src homology 2 domain containingtransforming protein 3, myeloperoxidase, lipocalin 2, cathepsin G,bactericidal/permeability-increasing protein, lactotransferrin (LTF),solute carrier family 22 member 2, methyltrarssferase like 7B, resistin.zinc finger protein 90, family with sequence similarity 46 member C,solute carrier family 7 member 5, aminolevulinate delta-synthase 2,5′-nucieotidase domain containing 2, LOC646021, G antigen 12F,LOC100133Q75, hypothetical protein FLJ23865, RST17329 Athersys RAGELibrary cDNA, hypothetical protein LOC339047 transcript variant 74, CD40molecule TNF receptor superfamily member 5 transcript variant 2,splicing factor 1 transcript variant 4, synaptophysin-like 1 transcriptvariant 1, selenoprotein P plasma 1 transcript variant. 1, cDNADKFZp779F041 1, LOC643Q37, SH3-domain GRB2-like (endophilin) interactingprotein 1, microRNA 940, PR domain containing 12′, IMAGE clone 15667343,LOC100128771, i.OC?323871, region containing hypothetical proteinLOC283970 transcript variant 2 LOC643943, hypothetical proteinLQCI001297Q5, LOC728522, regulator of G-protein signaling 20 transcriptvariant 1, kelch domain containing 1, LOCI 00134669, tripartitemotif-containing 34 transcript variant 2, ribosomal protein LlO-like,LOC100129929, LOC402377, LOC642073, zinc finger protein 461, creatinekinase mitochondrial IB, yy43f04.s1 Soares melanocyte 2NbHM cDNA clone,KIAA1045, ZNF788, primary neuroblastoma clone Nbla10527, processing ofprecursor 5 ribonuclease P/MRP subunit transcript variant 2, myelintranscription factor 1-like, neuroblastoma breakpoint: family member 7phosphoKpase C-lifce 1, LOC39 176 1, LOC646498, solute carrier family 7member 6 transcript variant 2, LOCI 00134648, neuroblastoma breakpointfamily member 1 transcript variant 16, absent in melanoma 1-like,EPS8-hke 2, angiopoietin-like 6, LOC645743, V-set and transmembranedomain containing 2A, chemokine binding protein 2, LOC647121, familywith sequence similarity 19 (chemokine (C-C motif)-like) member A2,gamma-glutamyltransferase light chain 1 transcript variant A, LOC439949,LOC653157, neurotrophin 3, LOC649686, zinc finger protein 830 (ZNF830),glycerol kinase 5, leueme-rieh repeat-containing G protein receptor 6transcript variant 3, small nucleolar RNA C/D box 13, cDNA FLJ1 1554 lisclone HEMBA1003037, zinc finger protein 485; and determining theexpression levels of the one or more biomarkers by comparing theexpression levels of the at least one or more biomarkers to at least oneinvariant control marker wherein an increase or decrease in the level ofexpression of one or more biomarkers as compared to the at least oneinvariant control marker is indicative of a lower respiratory infection.

Embodiments of the present invention include a method of treating alower respiratory infection in a subject, including the steps of:obtaining a biological sample from the subject; detecting expressionlevels of one or more biomarkers in the biological sample selected fromthe group consisting of Neutrophil defensin 1 precursor, Defensin alpha1, LOC65360G, defensin alpha 1B, Src homology 2 domain containingtransforming protein 3, myeloperoxidase, Upocalin 2, cathepsin G,bactericidaj/peiineabiiity-increasing protein{circumflex over ( )}lacMransferrin (LTF), solute carrier family 22 member 2,methyltransferase like 7B, resistin, zinc finger protein 90, family withsequence similarity 46 member C, solute carrier family 7 member 5,aminolevulinate delta-synthase 2, 5′-nucleotidase domain containing 2,LOC646021, G antigen 12F, LOCI 00133075, hypothetical protein FLJ23865,RST17329 Athersys RAGE Library cDNA, hypothetical protein LOC339047transcript variant 74 CD40 molecule INF receptor superfamily member 5transcript variant 2, splicing factor 1 transcript variant 4.synaptophysin-Hke 1 transcript variant 1, selenoprotein P plasma 1transcript variant 1, cDNA DKFZp779F041 1, LOC643037, SH3-domainGRB2-like (endophilin) interacting protein 1, microRNA 940, PR domaincontaining 12, IMAGE clone 15667343, LOC10012877L LOC732387, regioncontaining hypothetical protein LOC283970 transcript variant 2LOC643943, hypothetical protein LOC100129705, LOC728522, regulator ofG-protein signaling 20 transcript variant 1, kelch domain containing 1,LQC1Q0134669, tripartite motif-containing 34 transcript variant 2,ribosomal protein L10-like, LOC100129929, LOC402377, LOC642073, zincfinger protein 461, creatine kinase mitochondrial IB, yy43f04.s1 Soeresmelanocyte 2NbHM cDNA clone, KIAA 1045, ZNF788, primary neuroblastomaclone:Nblal 0527, processing of precursor 5 ribonuclease P/MRP subunittranscript variant 2, myelin transcription factor 1-like, neuroblastomabreakpoint family member 7, phospholipase C-like 1, LOC391761,LOC646498, solute carrier family 7 member 6 transcript variant 2, LOCI00134648, neuroblastoma breakpoint family member 1 transcript variant16, absent in melanoma 1-like, EPS8-like 2, angiopoietin-like 6,LOC645743, V-set and transmembrane domain containing 2A, chemokinebinding protein 2, LOC647121, family with sequence similarity 19(chemokine (C-C motif}-like) member A2, gamma-glutamyl transferase lightchain 1 transcript variant A, LOC439949, LOC653157, neurotrophin 3,LOC649686, zinc finger protein 830 (ZNF83G), glycerol kinase 5,leucine-rich repeat-containing G protein receptor 6 transcript variant3, small nucleolar RNA C/D box 13, cDNA FLJ11554 f₁s clone HEMBA1003037,zinc finger protein 485; determining an increase or decrease in theexpression levels of said one or more biomarkers by comparing theexpression levels of said one or more biomarkers to at least oneinvariant control marker wherein an increase or decrease of at leastthree-fold in, the level of expression of said at one or more biomarkersas compared to the at least one invariant control marker is indicativeof a lower respiratory infection; and treating the subject for a lowerrespiratory infection,

Embodiments of the present invention include a kit for use in diagnosinga lower respiratory infection in a subject comprising: agents thatspecifically bind one or more biomarkers selected from the groupconsisting of Neutrophil defeatism 1 precursor, Defensin alpha 1,LOC653600, defensin alpha IB, Src homology 2 domain containingtransforming protein 3, myeloperoxidase, lipocalin 2, cathepsin G,bacterieidal/permeability-increasing protein, lactotransferrin (LTF),solute carrier family 22 member 2, methyl transferase like 7B, resistin,zinc finger protein 90, family with sequence similarity 46 member C,solute carrier family 7 member 5, aminolevulinate delta-synthase 2,5′-nucleotidase domain containing 2, LQC646021, G antigen 12F, LOC100133075, hypothetical protein FLJ23865, RST 17329 Athersys RAGELibrary cDNA, hypothetical protein LOC339047 transcript variant 74, CD40molecule TNF receptor superfamily member 5 transcript variant 2,splicing factor 1 transcript variant 4, syaaptophysin-like 1 transcriptvariant, selenoprotein P plasma 1 transcript variant 1, cDNADKFZp779F0411, LOC643037, SH3-domain GRB2-like (endophilin) interactingprotein 1, microRNA 940, PR domain containing 12, IMAGE clone 15667343,LOC100128771, LOC732387, region containing, hypothetical proteinLOC283970 transcript variant 2 LOC643943, hypothetical protein LOCI 00129705, LOC728522, regulator of G-protein signaling 20 transcript variant1, kelch domain containing I, LOG 100 134669, tripaititemotif-containing 34 transcript variant 2, ribosoraal protein 1,10-like,LOCI 00 129929, LOC402377, LOC642073, zinc finger protein 461, creatinekinase mitochondrial 1B, yy43f04.si Soares melanocyte 2NbHM cDNA clone,KIAA 1045, ZNF788, primary neuroblastoma clone:Nblal0527. processing ofprecursor 5 ribonuclease P/MRP subunit transcript variant 2, myelintranscription factor 1-like, neuroblastoma breakpoint family member 1,phospholipase C-like LOC391761, LOC646498, solute carrier family 7member 6 transcript variant 2, LQC100134648, neuroblastoma breakpointfamily member 1 transcript variant 16, absent in melanoma 1-like,EPS8-like 2, angiopoietin-like 6, LOC645743, V-set and transmembranedomain containing 2A, chemoldne binding protein 2, LOC647121, familywith sequence similarity 19 (ehemokine (C-C motif)-like) member A2,gamma-glutamyltransferase light chain 1 transcript variant A, LOC439949,LOC653157, neurotrophin 3, LOC649686, zinc finger protein 830 (ZNF830),glycerol kinase 5, leucine-rich repeat-containing G protein receptor 6transcript variant 3, small nucleolar RNA C/D box 13, cDNA FLJ11554 fisclone HEMBA1003037, zinc finger protein 485; agents that specificallybind to at least one invariant control marker; a container for housingsaid agents; and instructions for use of the agents for determining anincrease or decrease in the expression levels of said one or morebiomarkers by comparing the expression levels of said one or morebiomarkers to said at least one invariant control marker wherein anincrease or decrease in the level of expression of said one or motebiomarkers as compared to the at, least one invariant control marker isindicative of a lower respiratory infection. In such embodiments, theagents that bind to the biomarkers and/or the invariant control markerbind to nucleotide residues or versions and/or to peptide versions,motifs or residues or amino acid residues of the biomarkers andinvariant control marker,

Embodiments of the present invention include a method of diagnosing alower respiratory infection in a subject comprising: obtaining abiological sample from said subject; and contacting the biologicalsample from said subject with a kit for use in diagnosing a lowerrespiratory infection in a subject.

Embodiments of the present invention include a method of treating alower respiratory infection in a subject comprising: obtaining abiological sample from said subject; contacting the biological samplefrom said subject with a kit for use in diagnosing a lower respiratoryinfection in a subject; and treating the subject for a lower respiratoryinfection by administering antibiotics to said subject, administeringantivirals to said subject, administering anti¬inflammatories to saidsubject, or a combination thereof,

BRIEF DESCRIPTION OF THE DRAWINGS

Further objectives and advantages will become apparent from aconsideration of the description, drawings, and examples.

FIG. 1 is a graph showing biomarker levels in suspected lowerrespiratory infection and/or pneumonia patients versus control subjects.

DETAILED DESCRIPTION

Some embodiments of the current invention are discussed, in detailbelow. In describing embodiments, specific terminology is employed forthe sake of clarity. However, the invention is not intended to belimited to the specific terminology so selected. A person skilled in therelevant art will recognize that other equivalent components can beemployed and other methods developed without departing from the broadconcepts of the current invention. All references cited anywhere in thisspecification, including the Background and Detailed Descriptionsections, are incorporated by reference as if each had been individuallyincorporated.

Some embodiments of the current invention include an innovative methodfor diagnosing infectious diseases. An example based on an embodiment ofthe current invention uses the example of pneumonia, with a bloodbiomarker test based on independently confirmed, genomicaliy-derivedmarkers, In some embodiments, two major innovations are employed: 1)instead of soluble markers, the person's own circulating ceils are usedas the ‘canary in the mine’ to sense the pathogen, and 2) solid-phasecapture of specific cells, combined with specific fluorochromes used tocreate a small, sensitive, inexpensive, and rapid assay for infections.

The genomic-scale RNA expression screens of circulating cells describedherein reveal, strong cellular markers of pulmonary infections. In someembodiments, the RNA biomarkers are detectable in a handheldnanophotonic assay of whole cells, and are also expressed as changes inthe proteins they encode (e.g. amount or activity). The biomarkersdescribed herein can be employed in any of a variety of conventionalassays. Although embodiments described herein, are directed to thedetection of pneumonia, the markers and assays can also be used todiagnose other types of infections, sand can be readily adapted, forother specific applications, such as appendicitis (e.g. caused by aninfection), CNS infections, parasitic infections, and any situationwhere the immune system detects a pathogen.

Some embodiments of the present invention include methods and kits forassessing and treating lower respiratory infections in a subject, andmore particularly to assessing and treating lower respiratory infectionsin a subject using the analysis of biomarkers isolated from the subject,

In some embodiments, the invention relates to a method of diagnosing alower respiratory infection in a subject, including the steps: obtaininga biological sample from the subject; detecting expression levels of oneor more biomarkers in the biological sample selected from the groupconsisting of Neutrophil defensin 1 precursor, Defensin alpha 1,LOC653600, defensin alpha 1B, Src homology 2 domain containingtransforming protein 3, myeloperoxidase, lipocalin 2, cathepsin G,bactericidal/permeability-increasing protein, iactotransferrin (LTF),solute carrier family 22 member 2, methyltransferase like 7B, resistin,zinc finger protein 90, family with sequence similarity 46 member C,solute carrier family 7 member 5, aminolevulinate delta-synthase 2,5′-nucleotidase domain containing 2, LOC646021, G antigen 12F, LOC100133075, hypothetical protein FLJ23865, RST17329 Athersys RAGE LibrarycDNA, hypothetical protein LOC339047 transcript variant 74, CD40molecule TNF receptor superfamily member 5 transcript variant 2,splicing factor 1 transcript variant 4, synaptophysm-like 1 transcriptvariant 1, selenoprotein P plasma 1 transcript variant 1, cDNADKFZp779F0411, LOC643037, SH3-domain GRB2-like (endopliilin) interactingprotein 1, microRNA 940, PR domain containing 12, IMAGE clone 15667343,LOC100128771, LOC732387, region containing hypothetical proteinLOC283970 transcript va{dot over (r)} ant 2 LOC643943, hypotheticalprotein LOC100 129705, LOC728522, regulator of G-protein signaling 20transcript variant 1, kelch domain containing 1, LOC100 134669,tripartite motif-containing 34 transcript variant 2, ribosomal proteinLlO-like, LOCI 00129929. LOC4Q2377, LOC642073, zinc linger protein 461,creatine kinase mitochondrial IB, yy43f04.s1 Soares melanocyte 2NbHMcDNA clone, KIAA1045, ZNF788, primary neuroblastoma clone:Nblal0527,processing of precursor 5 ribonuclease P/MRP subunit transcript variant2, myelin transcription factor 1-like, neuroblastoma breakpoint familymember 7, phosphoiipase C-like 1, LOC391761, LOC646498, solute carrierfamily 7 member 6 transcript variant. 2, LOC1001 34648, neuroblastomabreakpoint family member 1 transcript variant 16, absent in melanomaHike, RPS8-like 2, angiopoietin-like 6, LOC645743, V-set andtransmembrane domain containing 2A, chemokine binding protein 2.LOC647121, family with sequence similarity 19 (chemokine (C-Cmotif)-like) member A2, gamma-glutamyltransferase light chain 1transcript variant A, LOC439949, LOC653157, neurotrophin 3, LOC649686,zinc finger protein 830 (ZNF830), glycerol kinase 5, leucine-richrepeat-containing G protein receptor 6 transcript variant 3, smallnucleolar RNA C/D box 13, cDNA FIJ 1 1554 fis clone HEMBA1003037, zincfinger protein 485; and determining the expression levels of the one ormore biomarkers by comparing the expression levels of the one or morebiomarkers to at least one invariant control marker wherein an increaseor decrease in the level of expression of the one or more biomarkers ascompared to the at least one invariant control marker is indicative of alower respiratory infection.

In some embodiments, the invention relates to a method of diagnosing alower respiratory infection in a subject, including the steps: obtaininga biological sample from the subject; detecting expression levels of atleast three biomarkers in the biological sample selected from the groupconsisting of Neutrophil defensin 1 precursor, Defensin alpha 1,LOC653600, defensin alpha 1B, Sre homology 2 domain containingtransforming protein 3, myeloperoxidase, lipocalin 2, cathepsin G,bactencidal/permeability-increasing protein, lactotransferrin (LTF),solute carrier family 22 member 2, methyltransferase like 7B, resistin,zinc finger protein 90, family with sequence similarity 46 member C,solute carrier family 7 member 5, aminolevulinate delta-synthase 2,5′-nucleotidase domain containing 2, LOC646021, G antigen 12F,LOC100133075, hypothetical protein FLJ2386S, RST17329 Athersys RAGELibrary cDNA, hypothetical protein LOC339047 transcript variant 74, CD40molecule TNF receptor superfamily member 5 transcript variant 2,splicing factor 1 transcript variant 4, synaptophysin-like 1 transcriptvariant 1, selenoprotein P plasma 1 transcript variant 1, cDNADKFZp779F0411, LOC643037, SH3-domain GRB2-like (endophilin) interactingprotein 1, microRNA 940, PR domain containing 12, IMAGE clone 15667343,LOC10012877L LOC732387, region containing hypothetical, proteinLOC283970 transcript variant 2 LOC643943, hypothetical proteinLOC100129705, LOC728522, regulator of G-protein signaling 20 transcriptvariant 1, ketch domain containing 1, LOCI 00134669, tripartitemotif-containing 34 transcript variant 2, ribosomal protein LlO-like,LOG 100129929, LOC402377, LOC642073, zinc finger protein 461, creatinekinase mitochondrial 1B, yy43f04.s1 Soares melanocyte 2NbHM cDNA clone,KIAA1045, ZNF788, primary neuroblastoma clone:Nblal0527, processing ofprecursor 5 ribonuclease P/MRP subiinit transcript variant 2, myelintranscription factor 1-like, neuroblastoma breakpoint family member 7,phospholipase C-like 1, LOC391761, LOC646498, solute carrier family 7member 6 transcript variant 2, LOC100134648, neuroblastoma breakpointfamily member 1 transcript variant 16, absent in melanoma 1-like,EPS8-like 2, angiopoietin-like 6, LOC645743, V-set and transmembranedomain containing 2A, chemokine binding protein 2, LOC647121, familywith, sequence similarity 19 (chemokine (C-C motif)-like) member A2,gamma-glutamyltransferase light chain 1 transcript variant A, LOC439949,LOC653157, neurotrophin 3, LOC649686, zinc finger protein 830 (ZNF830),glycerol kinase 5, leucine-rich repeat-containing G protein receptor 6transcript variant 3, small nucleolar RNA C/D box 13, cDNA FLJ1 1554fits clone HEMBA1 003037, zinc finger protein 485; and determining theexpression levels of the at least three biomarkers by comparing theexpression levels of the at, least three biomarkers to at least oneinva{dot over (r)}iant control marker wherein an increase or decrease inthe level of expression of the at least three biomarkers as compared tothe at least one invariant control marker is indicative of a lowerrespiratory infection,

Some embodiments of the present invention relate to a method ofdiagnosing a lower respiratory infection in a subject comprisingcalculating a biomarker infection score from an increase or decrease inthe expression levels of one or more biomarkers and comparing saidbiomarker infection score to a control score.

Some embodiments of the present invention relate to a method ofdiagnosing a lower respiratory infection in a subject comprisingdetermining the expression levels of one or more biomarkers and at leastone invariant control marker by using a multivariate prediction model todetermine if a pattern of expression of said one or more biomarkers isindicative of a lower respiratory infection.

Some embodiments of the present invention relate to a method ofdiagnosing a lower respiratory infection in a subject, wherein a bloodsample is used as a biological sample,

Some embodiments of the present invention relate to a method ofdiagnosing a lower respiratory infection in a subject comprisingisolating immune cells from a blood sample,

Some embodiments of the present invention relate to a method ofdiagnosing a lower respiratory infection in a subject comprisingisolating neutrophils, T-cells, or a combination thereof from thesubject,

Some embodiments of the present invention relate to a method ofdiagnosing a lower respiratory infection in a subject comprisingdetecting expression levels of one or more biomarkers by measuring RNAlevels of the one or more biomarkers,

Some embodiments of the present invention relate to a method ofdiagnosing a lower respiratory infection in a subject comprisingmeasuring RNA levels of one or more biomarkers by usingfluorescently-labeled probes complementary to the one or morebiomarkers, a ligase-based assay, reverse transcriptase and polymerasechain reaction, RNA sequencing, or cDNA microarray.

Some embodiments of the present invention relate to a method ofdiagnosing a lower respiratory infection in a subject comprisingdetecting expression levels of one or more biomarkers selected from thegroup consisting of bactericidal/permeability-increasing protein,myeloperoxidase, resistin, G antigen 12F, CD40 molecule TNF receptorsuperfamily member 5 transcript variant 2 and splicing factor 1transcript variant 4.

Some embodiments of the present invention relate to a method ofdiagnosing a lower respiratory infection in a subject comprisingdetecting expression levels of one or more biomarkers selected from thegroup consisting of alkaline phosphatase liver isotbrm (ALPL),interleukin-S receptor-beta (IL8RB), and Defensin alpha 1.

Some embodiments of the present invention relate to a method ofdiagnosing a lower respiratory infection hi a subject comprisingdetecting expression levels of one or more biomarkers and at least oneinvariant control marker selected from the group consisting of Spi-Btranscription factor, protein phosphatase 1 regulatory subunit 21 (PPP1R21, KLRAQ1), mitogen-activated protein kinase kinase, kinase 7(MAP3K7, TAK1), olfactory receptor family 51 subfamily member 1 (OR51M1), BCL2 antagonist/killer (BAK 1), and adenosine deaminase,RNA-specific (ADAR1).

Some embodiments of the present invention include a method of treating alower respiratory infection in a subject, including the steps of:obtaining a biological sample from the subject; detecting expressionlevels of one or more biomarkers in the biological sample selected fromthe group consisting of Neutrophil defensin 1 precursor, Defensin alpha1, LOC653600, defensin alpha IB, Src homology 2 domain containingtransforming protein 3, myeloperoxidase, lipocalin 2. cathepsin G,bactericidal/permeability-increasing protein, lactotransferrin (LTF),solute carrier family 22 member 2, methyl transferase like 7B, resistin,zinc finger protein 90, family with sequence similarity 46 member C,solute carrier family 7 member 5, aminoleviilmate delta-synmase 2,5′-nucleotidase domain containing 2, LOC646021, G antigen 12F,LOC100133075, hypothetical protein FLJ23865, RST17329 Athersys RAGELibrary cDNA, hypothetical protein LOC339047 transcript variant 74, CD40molecule TNF receptor superfamily member 5 transcript variant 2,splicing factor 1 transcript variant 4, synaptophysin-like 1 transcriptvariant 1, selenoprotein P plasma 1 transcript variant 1, cDNADKFZp779F0411, LOC643037, SH3-domain GRB2-like (endophilin) interactingprotein 1, microRNA 940, PR domain containing 12, IMAGE clone 15667343,LOC100128771, LOC732387, region containing hypothetical proteinLOC283970 transcript variant 2 LOC643943, hypothetical protein LOCI00129705, LOC728522, regulator of G-protein signaling 20 transcriptvariant 1, kelch domain containing 1, LOG100134669, tripartitemotif-containing 34 transcript variant 2, ribosomal protein LlO-like,LOC100129929, LOC402377, LOC642073, zinc finger protein 461, creatinekinase mitochondrial IB, yy43fl)4.s1 Soares melanocyte 2NbHM cDNA clone,KIAA1045, ZNF788, primary neuroblastoma clone:Nblal0527, processing ofprecursor 5 ribonuclease P/MRP subunit transcript variant 2, myelintranscription factor 1-like, neuroblastoma breakpoint family member 7,phospholipase C-like 1, LOC391761, LOC646498, solute carrier family 7member 6 transcript variant 2, LOCI 00134648, neuroblastoma breakpointfamily member 1 transcript variant 16, absent in melanoma 1-like,EP88-like 2, angiopoietin-like 6, LOC645743, V-set and transmembranedomain containing 2A, chemokine binding protein 2, LOC647121, familywith sequence similarity 19 (chemokine (C-C motif)-like) member A2,gamma-glutamyltransferase light chain 1 transcript variant A, LOC439949,LOC653157, neurotrophin 3, LOC649686, zinc finger protein 830 (ZNF830),glycerol kinase 5, leucine-rich repeat-containing G protein receptor 6transcript variant 3, small nucleolar RNA C/D box 13, cDNA FIJI 1554 fisclone HEMBA1003037, zinc finger protein 485; determining an increase ordecrease in the expression levels of said one or more biomarkers bycomparing the expression levels of said one or more biomarkers to atleast one invariant control marker wherein an increase or decrease inthe level of expression of said one or more biomarkers as compared tothe at least one invariant control marker is indicative of a lowerrespiratory infection; and treating the subject for a lower respiratoryinfection.

Some embodiments of the present invention include a method of treating,a lower respiratory infection in a subject, including the steps of:obtaining a biological sample from the subject; detecting expressionlevels of at least three biomarkers in the biological sample selectedfrom the group consisting of Neutrophil defensin 1 precursor, Defensinalpha 1, LOC653600, defensin alpha IB, Src homology 2 domain containingtransforming protein 3, myeloperoxidase, lipocalin 2, cathepsin G,bactericidal/permeability-increasing protein, lactotransfer{dot over(r)} n (CIF), solute carrier family 22 member 2, methyltransferase like7B, resisting zinc finger protein 90, family with sequence similarity 46member C, solute carrier family 7 member 5, aminolevulmatedelta-synthase 2, 5′-nucieotidase domain containing 2, LOC646021, Gantigen 12F, LOCI 00133075, hypothetical protein FLJ23865, RST17329Athersys RAGE library cDNA, hypothetical protein LOC339047 transcriptvariant 74, CD40 molecule TNF receptor superfamily member 5 transcriptvariant 2, splicing factor 1 transcript variant 4, synaptophysin-like 1transcript variant 1, selenoprotein P plasma 1 transcript variant 1,cDNA DKFZp779F041 1, LOC643037, SH3-domain GRB2-like (endophilm)interacting protein 1, rnieroRNA 940, PR domain containing 12, IMAGEclone 15667343, LOC100128771, LOC732387, region containing hypotheticalprotein LOC283970 transcript variant 2 LOC643943, hypothetical proteinLOCI 00129705, LOC728522, regulator of G-protein signaling 20 transcriptvariant 1, kelch domain containing 1. LOC100134669, tripartitemotif-containing 34 transcript variant 2, ribosomal protein LI0-like,LOCI 00129929, LOC402377, LOC642073, zinc finger protein 461, creatinekinase mitochondrial IB, yy43f04.s1 Soares melanocyte 2NbHM cDNA clone,KIAA1045, ZNF788, primary neuroblastoma done:Nblal0527, processing ofprecursor 5 ribonuclease P/MRP subunit transcript variant 2, myelintranscription factor 1-like, neuroblastoma breakpoint family member 7,phospholipase 1, LOC391761, LOC646498, solute earner family 7 member 6transcript variant 2, LOC100 134648, neuroblastoma breakpoint familymember 1 transcript variant 16, absent in melanoma 1-like, EPS8-like 2,angiopoietin-like 6, LOC645743, V-set and transmembrane domaincontaining 2A, chernokine binding protein 2, LOC647121, family withsequence similarity 19 {chernokine (C-C motif)-like) member A2,gamma-glutamyltransterase light chain 1 transcript variant A, LOC439949,LOC653157, neurotrophin 3, LOC649686, zinc finger protein 830 (ZNF830),glycerol kinase 5, ieucme-rich repeat-containing G protein receptor 6transcript variant 3, small nucleolar RNA C/D box 13, cDNA FIJI 1554 fisclone HEMBA1003037, zinc finger protein 485; determining an increase ordecrease in the expression levels of said at least three biomarkers bycomparing the expression levels of said at least three biomarkers to atleast one invariant control marker wherein an increase or decrease inthe level of expression of said at least three biomarkers as compared tothe at least one invariant control marker is indicative of a lowerrespiratory infection; and treating the subject for a lower respiratoryinfection,

Some embodiments of the present invention relate to a method of treatinga lower respiratory infection in a subject, wherein treating the subjectfor a lower respiratory infection comprises administering antibiotics tosaid subject, administering antivirals to said subject, administeringanti-inflammatories to said subject, or a combination thereof.

Some embodiments of the present invention relate to a method of treatinga lower respiratory infection in a subject by calculating a biomarkerinfection score from said increase or decrease in the expression levelsof said one or more biomarkers and comparing said biomarker infectionscore to a control score.

Some embodiments of the present invention relate to a method of treatinga lower respiratory infection in a subject by determining an increase ordecrease in the expression levels of one or more biomarkers comprisingusing a multivariate prediction model to determine if a pattern ofexpression of said one or more biomarkers is indicative of a lowerrespiratory infection.

Some embodiments of the present invention relate to a method of treatinga lower respiratory infection in a subject by obtaining a blood samplefrom the subject and determining an increase or decrease in theexpression levels of one or more biomarkers.

Some embodiments of the present invention relate to a method of treatinga lower respiratory infection in a subject including isolating immunecells from a blood sample from the subject.

Some embodiments of the present invention relate to, a method oftreating, a lower respiratory infection in a subject comprisingisolating neutrophils, T-cells, or a combination thereof from thesubject.

Some embodiments of the present invention relate to a method of treatinga lower respiratory infection in a subject comprising detectingexpression levels of one or more biomarkers by measuring RNA levels ofsaid one or more biomarkers.

Some embodiments of the present invention relate to a method of treatinga lower respiratory infection in a subject comprising measuring RNAlevels of one or more biomarkers by using fluorescently-labeled probescomplementary to the said biomarkers, a ligase-based assay, reversetranscriptase and polymerase chain reaction, RNA sequencing or cDNAraicroarray.

Some embodiments of the present invention relate to a method of treatinga lower respiratory infection in a subject comprising determiningexpression levels of one or more biomarkers selected from the groupconsisting of bactericidal/permeability-increasing protein,myeloperoxidase, resistin, G antigen 12F, CD40 molecule TNF receptorsuperfamily member 5 transcript variant 2, and splicing factor 1transcript variant 4.

Some embodiments of the present invention relate to a method of treatinga lower respiratory infection in a subject comprising determiningexpression levels of one or more biomarkers selected from the groupconsisting of defensin alpha-1, alkaline phosphatase liver isoform(ALPL), interleukin-8 receptor beta (IL8RB).

Some embodiments of the present invention relate to a method to treatinga lower respiratory infection in a subject comprising determiningexpression levels of one or more biomarkers by comparing the expressionlevels of the one or more biomarkers to at least one invariant controlmarker selected from the group consisting of Spi-B transcription factor,protein phosphatase 1 regulatory subunit 21 (PPP1R21, KLRAQ1),mitogen-activated protein kinase kinase kinase 7 (MAP3K7, TAK1),olfactory receptor family 51 subfamily member 1 (OR51M1), BCL2antagonist/killer (BAK1), and adenosine deaminase, RNA-specific (ADAR1).

Some embodiments of the present invention include a kit for use indiagnosing lower respiratory infection in a subject comprising; agentsthat specifically bind one or more biomarkers selected from the groupconsisting of Neutrophil defensin 1 precursor, Defensin alpha 1,LOC653600, defensin alpha IB, Src homology 2 domain containingtransforming protein 3, myeloperoxidase, lipocalin 2, cathepsin G,bactericidal/permeability-increasing protein, lactotransferrin (LTF),solute carrier family 22 member 2, methyltransferase like 7B, resistin,zinc finger protein 90, family with sequence similarity 46 member C,solute carrier family 7 member 5, amino, evulmate delta-synmase 2,5′-nucleotidase domain containing 2, LOC646021, G antigen 12F, LOCI 00133075, hypothetical protein FLJ23865, RST17329 Athersys RAGE LibrarycDNA, hypothetical protein LOC339047 transcript variant 74, CD40molecule TNF receptor superfamily member 5 transcript variant 2,splicing factor 1 transcript variant 4, synaptophysin-like 1 transcriptvariant 1, selenoprotein P plasma 1 transcript variant 1, cDNADKFZp779F0411, LOC643037, SH3-domain GRB2-like (endophilin) interactingprotein 1, microRNA 940, PR domain containing 12 IMAGE clone 15667343,LOC100128771, LOC732387, region containing hypothetical proteinLOC283970 transcript variant 2 LOC643943, hypothetical proteinLOC100129705, LOC728522, regulator of G-protein signaling 20 transcriptvariant 1, kelch domain containing 1, LOC100134669, tripartitemotif-containing 34 transcript variant 2, ribosomal protein LlO-like,LOC100129929, LOC402377, LOC642073, zinc finger protein 461, creatinekinase mitochondrial IB, w43f04.s1 Soares melanocyte 2NbHM cDNA clone,KIAA1Q45, ZNF788, primary neuroblastoma clone:Nblai0527, processing ofprecursor 5 ribonuclease P/MRP subunit transcript variant 2, myelintranscription factor 1-like, neuroblastoma breakpoint family member 7,phospholipase C-like 1, LOC391761; LOC646498, solute carrier family 7member 6 transcript variant 2, LOG1001 34648, neuroblastoma breakpointfamily member 1 transcript variant 16, absent in melanoma 1-like,EPS8-like 2, angiopoietin-like 6, LOC645743, V-set and transmembranedomain containing 2A, chemokine binding protein 2, LOC647121, familywith sequence similarity 19 (chemokine (C-C motif)-like) member A2,gamma-glutamyltransferase light chain 1 transcript variant A, LOC439949,LOC653157, neurotrophin 3, LOC649686, zinc finger protein 830 (ZNF830),glycerol kinase leucine-ricb repeat-containing G protein receptor 6transcript variant 3, small nucleolar RNA C/D box 13, cDNA FIJI 1554 fisclone HEMBA1003037, zinc finger protein 485: agents that specificallybind to at least one invariant control marker; a container for housingsaid agents; and instructions for use of the agents for determining anincrease or decrease in the expression levels of said one or morebiomarkers by comparing the expression levels of said one or morebiomarkers to said at least one invariant control marker wherein anincrease or decrease in the level of expression of said one or morebiomarkers as compared to the at least one invariant control marker isindicative of a lower respiratory infection.

Some embodiments of the present invention include a kit for use indiagnosing a lower respiratory infection in a subject comprising: agentsthat specifically bind at least three biomarkers selected from the groupconsisting of Neutrophil defensin 1 precursor, Defensin alpha 1,LOC6S3600, defensin alpha IB, Src homology 2 domain containingtransforming protein 3, myeloperoxidase, lipocalin 2, cathepsin Gbactericidal/permeability-increasing protein, lactotransferrin (LTF),solute carrier family 22 member 2, methyltransferase like 7B, resistin,zinc finger protein 90, family with sequence similarity 46 member C,solute carrier family 7 member 5, aminolevulinate delta-synthase 2,5′-nucleotidase domain containing 2, LOC646021, G antigen 12F,LOC100133075, hypothetical protein FLJ23865, RST17329 Athersys RAGELibrary cDNA, hypothetical protein LOC339047 transcript variant 74, CD40molecule TNF receptor superfamily member 5 transcript variant 2,splicing factor 1 transcript variant 4, synaptophysin-like 1 transcriptvariant 1, selenoprotein P plasma 1 transcript variant 1, cDNADKFZp779F0411, LOC643037, SH3-domain GRB2-Sike (endophilin) interactingprotein 1, microRNA 940, PR domain containing 12, IMAGE clone 15667343,LOC100128771, LOC732387, region containing hypothetical proteinLOC283970 transcript variant 2 LOC643943, hypothetical proteinLOC100129705, LOC728522, regulator of G-protein signaling 20 transcriptvariant 1, kelch domain containing 1, LOG 100 134669, tripartitemotif-containing 34 transcript variant 2, ribosomal protein LlO-like,LOCI 00129929, LOC402377, LOC642073, zinc finger protein 461, creatinekinase mitochondrial IB, yy43f04.$1 Soares melanocyte 2NbHM cDNA clone,KIAA1045, ZNF788, primary neuroblastoma clone:Nbla 10527, processing ofprecursor 5 ribonuclease P/MRP subunit transcript variant. 2, myelintranscription factor 1-like, neuroblastoma breakpoint family member 7,phospholipase like 1, LOC391761, LOC646498, solute carrier family 7member 6 transcript variant 2, LOCI 00134648, neuroblastoma breakpointfamily member 1 transcript variant 16, absent in melanoma 1-like,EPS8-Kke 2, angiopoietin-like 6, LOC645743, V-set and transmembranedomain containing 2A, chemokine binding protein 2, LGC647121, familywith sequence similarity 19 (chemokine (C-C motif)-like) member A2,gamma-glutamyltransferase light, chain 1 transcript variant A,LOC439949, LOC653157, neurotropism 3, LOC649686, zinc finger protein 830(ZNF830), glycerol kinase 5, leucine-rich repeat-containing G proteinreceptor 6 transcript variant 3, small nucleolar RNA C/D box 13, cDNAFLJ11554 fis clone HEMBA1003037, zinc finger protein 485; agents thatspecifically bind to at least one invariant control marker; a containerfor housing said agents; and instructions for use of the agents fordetermining an increase or decrease in the expression levels of said atleast, three biomarkers by comparing the expression levels of said atleast three biomarkers to said at least one invariant control markerwherein an increase or decrease in the level of expression of said atleast three biomarkers as compared to the at least one invariant controlmarker is indicative of a lower respiratory infection.

Some embodiments of the present invention relate to a kit for use indiagnosing a lower respiratory infection in a subject comprisingpolynucleotides that specifically bind to RNA transcripts of one or morebiomarkers and of at least one invariant control marker.

Some embodiments of the present invention relate to a kit for use indiagnosing a lower respiratory infection in a subject comprisingpolynucleotides labeled with a detectable marker.

Some embodiments of the present invention relate to a kit for use indiagnosing a lower respiratory infection in a subject comprisingpolynucleotides that amplify polynucleotides encoding, one or morebiomarkers and at least one invariant control marker,

Some embodiments of the present invention relate to a kit for use indiagnosing a lower respiratory infection in a subject comprisinginstructions for calculating a biomarker infection score from anincrease or decrease in the expression levels of one or more biomarkersand comparing said biomarker infection score to a control score.

Some embodiments of the present invention relate to a kit for use indiagnosing a lower respiratory infection in a subject comprisinginstructions for determining an increase or decrease in the expressionlevels of one or more biomarkers by using a multivariate predictionmodel to determine if a pattern of expression of said one or morebiomarkers is indicative of a lower respiratory infection,

Some embodiments of the present invention relate to a kit for use indiagnosing lower respiratory infection in a subject comprising agentsthat specifically bind to one or more biomarkers selected from the groupconsisting of bactericidal/permeability-increasing protein,myeloperoxidase, resistin, G antigen 12F, CD40 molecule TNF receptorsuperfamily member 5 transcript variant 2, and splicing factor 1transcript variant 4,

Some embodiments of the present invention relate to a kit for use indiagnosing a lower respiratory infection in a subject comprising agentsthat specifically bind to defense alpha-i, alkaline phosphatase liverisoform (ALPL), interleukin-8 receptor beta (IL8RB).

Some embodiments of the present invention relate to a kit for use indiagnosing a lower respiratory infection in a subject comprising agentsthat specifically bind to at least one invariant control marker selectedfrom the group consisting of actin-beta (ACTB), Spi-B transcriptionfactor, protein phosphatase 1 regulatory subunit 21 (PPP1R21, KLRAQ1),mitogen-activated protein kinase kinase kinase 7 (MAP3K7, TAK1),olfactory receptor family 51 subfamily member 1 (OR51M1), BCL2antagonist/killer (BAK1), and adenosine deaminase, RN A-specific(ADAR1).

Some embodiments of the present invention relate to a kit for use indiagnosing a lower respiratory infection in a subject comprisingreagents for fluorescently-labeled probes complementary to the one ormore biomarkers, a ligase-based assay, reverse transcriptase andpolymerase chain reaction, RNA sequencing or cDNA microarray.

Some embodiments of the present invention include a method of diagnosinga lower respiratory infection in a subject comprising: obtaining abiological sample from said subject; and contacting the biologicalsample from said subject with a kit for use in diagnosing a lowerrespiratory infection in a subject,

Some embodiments of the present invention include a method of treating alower respiratory infection in a subject comprising: obtaining abiological sample from said subject; contacting the biological samplefrom said subject with a kit for use in diagnosing a lower respiratoryinfection in a subject; and treating the subject for a lower respiratoryinfection by administering antibiotics to said subject, administeringantivirals to said subject, administering anti¬inflammatories to saidsubject, or a combination thereof.

Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below,

As used herein, the singular forms “a”, “an”, and “the” include pluralforms unless the context clearly dictates otherwise. Thus, for example,reference to “a binding agent” includes reference to more than onebinding agent.

The terms “diagnostic” and “diagnosis” refer to identifying the presenceor nature of a pathologic condition and includes identifying patientswho are at risk of developing a specific disease or disorder. Diagnosticmethods differ in their sensitivity and specificity. The “sensitivity”of a diagnostic assay is the percentage of diseased individuals who testpositive (percent of “true positives”). Diseased individuals notdetected by the assay are “false negatives.” Subjects who are notdiseased and who test negative in the assay, are termed “truenegatives.” The “specificity” of a diagnostic assay is 1 minus the falsepositive rate, where the “false positive” rate is defined as theproportion of those without the disease who test positive. While aparticular diagnostic method may not provide a definitive diagnosis of acondition, it suffices if the method provides a positive indication thataids in diagnosis.

The terms “detection”, “detecting” and the like, may be used in thecontext of detecting bio markers, or of detecting a disease or disorder(e.g., when positive assay results are obtained). In the latter context,“detecting” and “diagnosing” are considered synonymous.

The terms “subject”, “patient” or “individual” generally refer to ahuman, although the methods of the invention are not limited 0 humans,and should be useful in other mammals (e.g., eats, dogs, etc).

“Sample” is used herein in its broadest sense. A sample may comprise abodily fluid including blood, serum, plasma, tears, sputum, broncheolarlavage, aqueous and vitreous humor, spinal fluid, urine, and saliva; asoluble fraction of a cell or tissue preparation, or media in whichcells were grown. Means of obtaining suitable biological samples areknown to those of skill in the art.

An “antibody” is an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, etc., through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. As used herein, the term is used in the broadest sense andencompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such, as Fab, Fab′, F(ab′)2, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,hybrid, antibodies, fusion proteins comprising an antibody portion, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody may be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, igG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit. structures andthree-dimensional configurations. Antibodies may be naked or conjugatedto other molecules such as toxins, radioisotopes, enzymes,fluorochromes. etc.

The term “antibody fragments” refers to a portion of an intact antibody.Examples of antibody fragments include, but are not limited to, linearantibodies; single-chain antibody molecules; Fc or Fc′ peptides, Fab andFab fragments, and multispecific antibodies formed from antibodyfragments.

“Hybrid antibodies” are immunoglobulin molecules in which pairs of heavyand light chains from antibodies with different antigenic determinantregions are assembled together so that two different epitopes or twodifferent antigens may be recognized and bound by the resultingtetramer.

“isolated” in regard to cells, refers to a cell that is removed from itsnatural environment and that is isolated or separated, and is at leastabout 30%, 50%, 75%, and 90% free from other cells with which it isnaturally present, but which lack the marker based on which the cellswere isolated.

For use in the diagnostic and therapeutic applications described herein,kits are also within the scope of some embodiments of the currentinvention. Such kits can comprise a carrier, package or container thatis compartmentalized to receive one or more containers such as vials,tubes, and the like, each of the containers) comprising one of theseparate elements to be used in the method. For example, the containers)can comprise a probe that is or can be detectably labeled. The probe canbe an antibody or polynucleotide specific for a biomarker of interest.Alternatively, the kit can comprise a mass spectrometry (MS) probe. Thekit can also include containers containing nucleotide(s) foramplification or silencing of a target nucleic acid sequence, and/or acontainer comprising a reporter, such as a biotin-binding protein, e.g.,avidin or streptavidin, bound to a detectable label, e.g., an enzymatic,florescent, or radioisotope label. The kit can include Fall or part ofthe amino acid sequence of the biomarker, or a nucleic acid moleculethat encodes such amino acid sequences.

A kit according to an embodiment of the current invention may comprisethe container described above and one or more other containerscomprising materials desirable from a commercial and user standpoint,including buffers, diluents, filters, needles, syringes, and packageinserts with instructions for use. In addition, a label can be providedon the container to indicate that the composition is used for a specifictherapeutic or non-therapeutic application, and can also indicatedirections for either in vivo or in vitro use, such as those describedabove. Directions and or other information can also be included on aninsert that is included with the kit.

Polynucleotides may be prepared using any of a variety of techniquesknown in the art. The polynucleotide sequences selected as probes (andbind to the biomarkers of interest) should be sufficiently long andsufficiently unambiguous that false positives are minimized. Thepolynucleotide is preferably labeled such that it can be detected uponhybridization to DNA and/or RNA in the assay being screened. Methods oflabeling are well known in the art, and include the use of radiolabels,such as 32P-labeled ATP, biotmylation, fluorescent groups or enzymelabeling. Hybridization conditions, including moderate stringency andhigh stringency, are well known in the art.

Polynucleotide variants may generally be prepared by any method known inthe art, including chemical synthesis by, for example, solid phasephosphoramidite chemical synthesis, Modifications in, a polynucleotidesequence may also be introduced using standard mutagenesis techniques,such as oligonucleotide-directed site-specific mutagenesis.Alternatively, DNA and RNA molecules may be generated in vitro or invivo. Certain portions may be used to prepare an encoded polypeptide,

Any polynucleotide may be further modified to increase stability in vivoand/or in vitro for improved activity and/or storage. Possiblemodifications include, but are not limited to, the addition of flankingsequences at the 5′ and/or 3′ ends; the use of phosphorothioate or 2′O-methyl rather than phosphodiesterase linkages in the backbone; and/orthe inclusion of nonuraditional bases such as inosine, queosine andwybutosine, as well as acetyl-methyl-, thio- and other modified forms ofadenine, cytidine, guanine, thymine and uridine.

Polynucleotides and/or antibodies specific to biomarkers of interest canbe conjugated to detectable markers to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a biolummescent compound, chemiluminescentcompound, a metal chelator or, an enzyme. A second molecule forconjugation can be selected in accordance with the intended use. Forexample, for therapeutic use, the second molecule can be a toxin ortherapeutic agent. Further, bi-specific antibodies specific for two ormore biomarkers may be generated using methods generally known in theart. Homodimeric antibodies may also be generated by cross-linkingtechniques known in the art.

EXAMPLES

The following examples help explain some concepts of the currentinvention. However, the general concepts of the current invention arenot limited to the particular examples.

Example 1: Blood Biomarkers for Respiratory Infections

The following factors are taken into consideration in this embodiment ofthe present invention:

1) Sample: In some embodiments, whole blood is the primary type ofsample, in which the whole blood is drawn into a suitable preservativeknown to stabilize and protect the RNA. Blood drawn by venipuncture,in-dwelling catheter, or finger stick, are recognized and reliablemethods of obtaining a blood sample from a human or other subject.

2) Time/Cost. Time is a major design driver. Prolonged analytical timea) delays treatment, b) requires revisit by the sick patient, and c)increases exposures to family and other patients. The use of RNA and/orcell-based systems could dramatically reduce time sand costs because thevolume of reagents in a microchamber can be extremely efficient and thetime of quantitation can be reduced to hours or minutes, depending onthe technology employed.

3) Sensitivity. In many infections, including LRI, and especiallyappendicitis (e.g. resulting from infection), sinus infections, andbrain infections, the pathogen may never circulate at detectable levelsin blood. In many cases, bacteria can form ‘biofilms’, which can linethe appendix, lungs, or sinuses and cause extreme inflammation withoutever circulating in blood. However, the presently claimed method takesadvantage of the fact that the blood will contain immune cells that havepassed close, enough b become activated by pathogen-derived diffusiblesignals, such as endotoxin and butyrate.

4) Selectivity, A cell-based system can distinguish viral from bacterialinfections because the cellular-systems that respond to viruses andbacteria are quite different. Viral markers as well as bacterial markerscan be used in embodiments of the invention. Clearly, the first questionis really whether the person has an infection, versus an inflammatoryreaction to allergens, a persistent inflammatory condition, such asbronchitis, asthma, or chronic obstructive pulmonary disease (COPD),which are treated very differently from an infection. By ruling out aninfection, the doctor can decide, whether antihistamines for allergy, orsteroids for bronchitis are more appropriate.

Identification of Biomarkers in Blood.

Using genomic-scale RNA transcript profiling, 79 RNA markers in bloodwere identified. Any of these (alone or in combination with other of thebiomarkers) constitute a predictive biomarker of infection. Thesebiomarkers are used in any of a variety of conventional test formats,such as measuring RNA level, protein level, or protein activity.

Methods: A total of >270 patients presenting to the GW EmergencyDepartment were consented for a blood sample by venipuncture under anIRB-approved protocol. Their clinical course was unaffected by studyprocedures. Based on the final diagnosis, subsets of patients wereanalyzed by advanced genomic-scale RNA expression profiling of theirblood using Illumina BeadChip Arrays (v12.4). Expression profiles frompatients with acute appendicitis (APP), lower respiratory infection(LRI), or a presumably non-infectious control, abdominal hernia (HER),were compared to identify differentially expressed genes (DEG). Thestudies herein are focused on the LRI patients (n−5) compared to APP(n=9) and HER (η{circumflex over ( )}M), in which APP and HER are pooledto form a Control group (Con),

Targets: The gene expression profiling reported herein revealed 79transcripts (16 increased, 63 decreased) that were changed more than3-fold at an uncorrected p value of <0.001 (Table 1). Furthermore, 11 ofthese transcripts were elevated more than 6-fold and 51 decreased in LRIby more than 6-fold, providing good signal/noise ratio for a biomarker.Published microarray analysis of whole blood RNA from chronicobstructive pulmonary disease (COPD) patients has identified some of themarkers described herein. However, their levels were associated with theprimary endpoint of ICU vs non-ICU status of the patient, and not.specifically the presence of a respiratory infection (ALmansa 2012). Inembodiments of the present invention, markers that are specificallyassociated with respiratory infections are employed in order to detectinfections in a patient without regard b ICU status, but rather asgeneral guide to a physician in determining therapy. In some embodimentsof the present invention, the previously identified markers are used inconjunction with one or more new markers that are identified herein.

Examples of LRI biomarkers in blood: (A full list of the 79 biomarkersis found in ‘fable 1 below)

Transcripts elevated in LRI; Of the 79 transcripts, 16 transcripts wereincreased in LRI, and several of these transcripts have known proteins,some of which have been associated with the response to infection.However, the levels of these RNAs have not been shown to be diagnosticof infection. Exemplary markers that are used in embodiments of theinvention include;

Bactericidaypenneability-increasing protein (BPI), increased >7-fold inLRI, is known to b e elevated in patients with LRI (Lange 2013).Patients with severe sepsis show elevated plasma levels of BPI protein,defensin A1 (DEFA1) protein, and lactoferrin (LTV) protein (Berkested t2010). In some embodiments of the present invention, the previouslyidentified markers are used in conjunction with one or more new markersthat are identified herein.

Myeloperoxidase (MPQ), increased >9-fold in LRI, is a neutrophil granuleprotein, which works with NADPH oxidase to convert H202 to hypochlorousacid (bleach), which is a potent mierobtocidal agent. MPO generatesbleach in high concentrations within the phagosome that engulfs bacteriainto the neutrophil. While MPO mRNA is induced in LRI patients, secretedMPO protein in plasma is complicated due to specific neutralization ofMPO in plasma by agents such as cerruloplasmin, which limits the utilitya simple MPO activity test on plasma. Thus, cellular levels of MPO mRNAor protein are likely useful biomarkers for diagnostic tests.

Resistin (RETN), increased −6-fold in LRI, is stored in neutrophilgranules and released upon neutrophil activation, along with lactoferrinand CR3/CDllb (Bostrom 2009). Plasma levels of resistin, N GAL, and IL-8distinguished uncomplicated sepsis from septic shock (Macdonaki 2014).Resistin protein is elevated in septic patients, and released fromneutrophils by streptococcal cell wall (Johansson 2009). Resistin RNAand protein are principally in neutrophils, and to a lesser degreemonocytes. Resistin RNA expression is induced by Hpopolysaccharide (LPS)in neutrophils and U937 (Kunnari AM 2009 19445973), However, RETN hasnot been previously shown to be a predictive marker of infection (e.g.in humans),

Defensins (DEFA1B, HNP-L LOC653600) are a group of small peptides thatare intrinsically involved in the host defense against pathogens.Typically contained within the azurophilic neutrophil granules, theseproteins can directly attack bacteria, and potentially viruses, whenthey internalized within a host immune cell such as the neutrophil. Thespecific defensins identified in our microarray screen are multipletranscripts which derive from the DEFA1/3 locus in the genome at thelocation chr8:6,977,650-7,018,500 in the hg38 genome build, A variety oftranscripts and proteins are made from this locus which are categorizedas DEFA1 or DEFA3 depending on the usage of the included exons. For thepurposes of the present invention, any transcripts derived from thislocus may be suitable for diagnosis, and the DEFA1 version of the mRNAis used for demonstration of the utility, but is not limiting inpractice of the invention,

Transcripts which are decreased in LRI:

GAGE12F transcript is decreased ˜17-fold in patients with LRL Nobiological or physiological function has been described for GAGE12F,and, it has not been shown previously to be a biomarker for infection,

CD40 transcript is >12-fold decreased in patients with LRL GD40 is awell-known protein that is grouped into the Tumor Necrosis Factor (TNF)receptor superfamily. However, decreased mRNA levels of CD40 have notbeen shown previously to be correlated with LRI or any other infection,

Splicing factor 1 (SF1) transcript variant 4, is decreased >12-fold inpatients with LRI, SF1 is a well-known protein involved in regulatingthe splicing of certain transcripts in diverse settings. However,decreased mRNA levels of SF1 (e.g. of transcript variant 4) have notbeen shown previously to be correlated with bacterial or viralinfection, e.g. of humans,

Invariant transcripts to be used as controls:

There are numerous transcripts that do not change between groups, andthese can be used as ‘normalizing’ genes (controls) to compensate forhandling and technical errors that might affect a particular sample.Examples of invariant gene transcripts include KLRAQ1, MAP3K7, OR51M1,BAK1, and ADAR1, although this list is not meant to be exclusive ofother invariant transcripts.

Calculation of the Infection Score:

The normalized signal values (SV) of 3 increased transcripts (RSTN, BPi,MPO) are added together and divided by the sum of the 3 invariant SVs,Likewise, the 3 decreased transcripts (GAGE12F, CD40, SF1) are addedtogether and divided by the sum of 3 invariant transcripts. In oneembodiment, ÷12.5 fold is added to [−14 fold] to create a score of 26.5,there would thus be a normal range and escalating probabilities ofinfection.

TABLE 1 Differentially expressed gene transcripts relevant to lowerrespiratory infection. ProbeID p-value FC (abs) Con LRI DEFINITION770400 3.61E−04 17.78 UP 0.43 4.58 Neutrophil defensin 1 precursor(HNP- 1) (HP-1) (HP1) (Defensin, alpha 1) (LOC653600), mRNA. 71501708.79E−04 15.79 UP 2.62 6.60 defensin, alpha 1B (DEFA1B), mRNA. 60406897.25E−04 9.32 UP −5.04 −1.82 SHC (Src homology 2 domain containing)transforming protein 3 (SHC3), mRNA. 3520601 8.41E−05 9.29 UP −0.31 2.91myeloperoxidase (MPO), nuclear gene encoding mitochondrial protein,mRNA. 4390398 2.40E−04 8.80 UP 3.04 6.18 lipocalin 2 (LCN2), mRNA.1500735 8.77E−04 7.95 UP 0.08 3.07 cathepsin G (CTSG), mRNA. 42007464.41E−04 7.48 UP 0.20 3.11 bactericidal/permeability-increasing protein(BPI), mRNA. 5550367 7.76E−04 6.98 UP 0.58 3.38 lactotransferrin (LTF),mRNA. 2340156 2.69E−04 6.85 UP −6.14 −3.37 solute carrier family 22(organic cation transporter), member 2 (SLC22A2), mRNA. 5310142 3.33E−046.60 UP −1.66 1.06 methyltransferase like 7B (METTL7B), mRNA. 46101299.27E−04 6.24 UP 0.86 3.50 resistin (RETN), mRNA. 5360427 6.81E−04 5.52UP −5.55 −3.08 zinc finger protein 90 (HTF9) (ZNF90), mRNA. 68603478.48E−05 3.93 UP 4.82 6.80 family with sequence similarity 46, member C(FAM46C), mRNA. 270152 2.80E−04 3.38 UP 1.57 3.33 solute carrier family7 (cationic amino acid transporter, y+ system), member 5 (SLC7A5), mRNA.5270338 8.32E−04 3.19 UP −1.00 0.68 aminolevulinate, delta-, synthase 2(ALAS2), nuclear gene encoding mitochondrial protein, transcript variant3, mRNA. 6840301 8.42E−04 3.13 UP −0.40 1.25 5′-nucleotidase domaincontaining 2 (NT5DC2), mRNA. 4060551 5.30E−07 19.07 Down −2.12 −6.37PREDICTED: similar to hCG1774990 (LOC646021), mRNA. 6620326 1.00E−0417.03 Down −2.28 −6.37 G antigen 12F (GAGE12F), mRNA. 990170 1.14E−0515.29 Down −1.09 −5.03 PREDICTED: misc_RNA (LOC100133075), miscRNA.4880280 4.09E−05 14.11 Down −2.55 −6.37 PREDICTED: hypothetical proteinFLJ23865 (FLJ23865), mRNA. 430162 4.06E−04 13.26 Down −2.64 −6.37RST17329 Athersys RAGE Library cDNA, mRNA sequence 1240382 1.11E−0413.03 Down −1.56 −5.26 PREDICTED: hypothetical protein LOC339047,transcript variant 74 (LOC339047), mRNA. 3370138 1.25E−04 12.53 Down−0.82 −4.47 CD40 molecule, TNF receptor superfamily member 5 (CD40),transcript variant 2, mRNA. 3610743 4.35E−04 12.30 Down 0.16 −3.46splicing factor 1 (SF1), transcript variant 4, mRNA. 5340224 5.34E−0511.75 Down −2.81 −6.37 synaptophysin-like 1 (SYPL1), transcript variant1, mRNA. 4280678 3.15E−04 11.25 Down −2.24 −5.73 selenoprotein P,plasma, 1 (SEPP1), transcript variant 1, mRNA. 6350093 3.87E−04 11.08Down −1.57 −5.04 mRNA; cDNA DKFZp779F0411 (from clone DKFZp779F0411)5390717 5.01E−05 10.87 Down −1.40 −4.85 PREDICTED: similar to hCG1730248(LOC643037), mRNA. 6380239 7.33E−04 10.82 Down −2.73 −6.17 SH3-domainGRB2-like (endophilin) interacting protein 1 (SGIP1), mRNA. 58604844.55E−05 10.69 Down −1.65 −5.07 microRNA 940 (MIR940), microRNA. 65806194.20E−04 10.68 Down −2.95 −6.37 PR domain containing 12 (PRDM12), mRNA.5810113 8.74E−04 10.59 Down −2.76 −6.17 oo20c08.x1Soares_NSF_F8_9W_OT_PA_P_S1 cDNA clone IMAGE: 1566734 3, mRNA sequence4390528 4.16E−04 10.26 −1.83 −5.19 PREDICTED: misc_RNA (LOC100128771),miscRNA. 5890598 3.41E−05 10.19 Down −2.18 −5.53 PREDICTED; similar toring finger protein 18 (LQC732387), mRNA. 6770068 2.91E−04 9.77 Down−2.88 −6.17 PREDICTED: region containing hypothetical protein LOC283970;similar to nuclear pore complex interacting protein, transcript variant2 (LOC643943), mRNA. 7570431 1.47E−04 9.50 Down −1.37 −4.62 PREDICTED:hypothetical protein LOC100129705 (LOC100129705), mRNA. 2650128 9.05E−059.40 Down −2.47 −5.70 PREDICTED: misc_RNA (LOC728522), miscRNA. 64004032.12E−04 9.27 Down −2.36 −5.57 regulator of G-protein signaling 20(RG820), transcript variant 1, mRNA. 2190100 9.82E−04 9.11 Down −1.83−5.02 keich domain containing 1 (KLHDC 1), mRNA. 6650192 7.74E−04 9.01Down −2.02 −5.19 PREDICTED: misc_RNA (LOC100134669), miscRNA, 24503006.13E−04 8.98 Down −3.20 −6.37 tripartite motif-containing 34 (TRIM34),transcript variant 2, mRNA. 5050072 3.85E−05 8.96 Down −1.43 −4.59ribosomal protein LlO-like (RPLIOL), mRNA. 4050482 7.43E−04 8.70 Down−2.47 −5.59 PREDICTED: similar to Fanconi anemia complementation groDOWND2 protein (LOC100129929), mRNA. 770110 5.48E−04 8.61 Down −2.12 −5.23PREDICTED: similar to UDP- Gal: betaGicNAc beta 1,3-galactosyltransferase, polypeptide 4 (LOC402377), mRNA. 3060520 7.72E−048.60 Down −0.20 −3.30 PREDICTED: similar to MHC class II antigen(LOC642073), mRNA. 1820474 5.80E−04 8.36 Down −2.47 −5.53 zinc fingerprotein 461 (ZNF461), mRNA. 5570021 4.57E−04 8.31 Down −2.60 −5.65creatine kinase, mitochondrial 1B (CKMT1 B), nuclear gene encodingmitochondrial protein, mRNA. 1010072 3.14E−04 8.16 Down −1.71 −4.74yy43ii)4.si Scares melanocyte 2NbHM cDNA clone IMAGE; 274015 3, mRNAsequence 2100162 9.49E−04 8.11 Down −2.06 −5.08 KIAA1045 (KÏAA 1045),mRNA. 5810682 1.58E−04 8.09 Down −1.87 −4.89 PREDICTED: misc_RNA(ZNF788), miscRNA, 6370228 6.94E−05 8.04 Down 0.09 −2.92 primaryneuroblastoma cDNA, clone: Nbla10527, full insert sequence 65504843.42E−04 8.03 Down −1.97 −4.97 processing of precursor 5, ribonucleaseP/MRP subunit (S. cerevisiae) (POPS), transcript variant 2, mRNA.2190358 2.25E−04 7.86 Down −1.94 −4.92 myelin transcription factor1-like (MYTI L), mRNA. 4590037 1.10E−04 7.81 Down −1.39 −4.36neuroblastoma breakpoint family, member 7 (NBPF7), mRNA. 21003566.42E−04 7.75 Down −1.70 −4.65 phospho!ipase C-like 1 (PLCL1), mRNA.6770452 2.13E−04 7.52 Down −1.89 −4.80 PREDICTED: similar to hCGl 809904(LOC391 761), mRNA. 5810768 1.78E−04 7.51 Down −1.36 −4.27 PREDICTED:hypothetical LOC646498 (LOC646498), mRNA. 6380364 4.53E−04 7.46 Down−1.27 −4.17 solute carrier family 7 (cationic amino acid transporter, y+system), member 6 (SLC7A6), transcript variant 2, mRNA. 3940750 1.13E−047.42 Down −1.91 −4.81 PREDICTED: similar to hCG2024 106, transcriptvariant 1 (LOC100134648), mRNA. 7380161 2.21E−04 7.27 Down −3.11 −5.97PREDICTED: neuroblastoma breakpoint family, member 1, transcript variant16 (NBPF1), mRNA. 4220376 2.06E−04 7.23 Down −2.11 −4.97 absent inmelanoma 1-like (AIM1L), mRNA. 5270603 4.32E−04 7.21 Down −1.50 −4.35EPS8-like 2 (EPS8L2), mRNA. XM_943956 XM_943960 XM_943963 XM_9439665050707 1.55E−04 7.19 Down −1.09 −3.93 angiopoietin-like 6 (ANGPTL6),mRNA. 4850110 4.74E−04 6.73 Down −2.09 −4.84 PREDICTED: hypotheticalprotein LOC645743 (LQC645743), mRNA. 5870091 6.14E−04 6.59 Down −2.03−4.75 V-set and transmembrane domain containing 2A (V STM2A), mRNA.460504 2.31E−04 6.50 Down −2.14 −4.84 chemokine binding protein 2(CCBP2), mRNA. 6350445 6.13E−04 6.20 Down −1.44 −4.07 PREDICTED: similarto embigin homolog (LOC647121), mRNA. 3520114 9.96E−04 5.71 Down −1.32−3.84 family with sequence similarity 19 (chemokine (C-C motitl-like),member A2 (FAM19A2), mRNA. 60735 5.14E−04 5.55 Down −2.64 −5.12gamma-glutamyltransferase light chain 1 (GGTLC1), transcript variant A,mRNA. 4560088 6.52E−04 5.54 Down 1.03 −1.44 PREDICTED: hypothetical genesDOWNported by AY007155 (LOC439949), mRNA. 4050132 6.18E−04 5.49 Down0.55 −1.91 PREDICTED: similar to Iduronate 2- sulfatase precursor(Alpha-L-iduronate sulfate sulfatase) (Idursulfase) (LOC653157), mRNA.21540441 6.28E−04 4.92 Down −1.75 −4.05 neurotrophin 3 (NTF3), mRNA.6020553 8.44E−04 4.67 Down −1.17 −3.39 PREDICTED: similar to Neuronalacetylcholine receptor protein, beta-4 subunit precursor (LOC649686),mRNA. 2320593 3.73E−04 4.23 Down −0.61 −2.69 zinc finger protein 830(ZNF830), mRNA. 6180543 6.30E−04 4.04 Down −0.51 −2.53 glycerol kinase 5(putative) (GK5), mRNA. 3610154 1.53E−04 4.00 Down −0.69 −2.69leucine-rich repeat-containing G protein- coDOWNled receptor 6 (LGR6),transcript variant 3, mRNA. 7210035 8.97E−04 3.60 Down 1.05 −0.80 smallnucleolar RNA, C/D box 13 (SNORD13), small nucleolar RNA. 52600211.18E−04 3.44 Down −0.66 −2.44 cDNA FLJ11554 fis, clone HEMBA1003037670279 1.34E−05 3.38 Down −0.49 −2.25 zinc finger protein 485 (ZNF485),mRNA.Table Headers:PROBEJD refers to the specific cDNA probe sequences used to detect theRNA transcript as described by the manufacturer of the Illumina BeadChip version 12.4.P value refers to the t-test probability that the two groups of samplesare actually derived from the same distribution of variance, A smallervalue indicates a greater chance that the expression levels in twogroups are significantly different in magnitude,FC (abs) refers to ‘fold change’ in absolute terms, i.e. corrected forthe log 2 expression levels in the subsequent columns, which is therelative change in expression between groups.Con refers to the control group, and LRI refers to the lower respiratoryinfection group, where the values are normalized expression valuesexpressed on log 2 scale. Thus, if Con is I and LR.1 is 3, then thedifference is 2²=4 fold change (FC)DEFINITION is the formal name of the transcript as described in Genbankor Refseq, as related by the manufacturer's library file for themicroarray.SYMBOL refers to the official gene symbol abbreviation, when available.The Genbank or Refseq numbers were obtained from NIH databases and theIllumina library file for the microarray. A given transcript can havemultiple Genbank identifiers reflecting different studies thatindependently observed the sequence is a specific context.

Additional information on the biomarkers from Table 1, including thegene symbol and synonyms appear in Table 2 below,

Homo sapiens primary neuroblastoma cDNA, clone:Nbla10527, full insertsequence: AB074162. pleckstrin homology domain interacting protein:AJ303102 AL049321 CH471051 BX537762 AF310250 BI548935 AL450327 BC137488BC021905 AK127816 BC008909 AK000712 AL161957 NM_017934 CR600369 BC036479BC064611 BC081569 DQ924532 AK057039 DA500781 AK075124 AL356776 BC144670. oo20c08.xl Soares_NSF_F8_9W_OT_PA_P_S1 Homo sapiens cDNA cloneIMAGE: 1566734 3, mRNA sequence: HS.134230. Homo sapiens KIAA1045(KIAA1045), mRNA: NM_015297. Homo sapiens kelch domain containing 1(KLHDC1), mRNA: BC143597 AK127202 AK098735 BC143596 AL591767 NM_172193BC101595 BC031270 BC101597 AF111806 AL833437 CH471078. Homo sapienslipocalin 2 (LCN2), mRNA: AK316217 N79823 CR542092 CH471090 BP272828X99133 EU644752 X83006 S75256 BX644845 BC033089 NM_005564 CA454137AL590708 AW778875 BM977724 AK301694 BF354583. Homo sapiens leucine-richrepeat-containing G protein-coupled receptor 6 (LGR6), transcriptvariant 3, mRNA: NM_001017404 BC047905 NM_001017403 AK123055 AK027377BM699180 AF088074 CH471067 AY358119 AF190501 CQ840811 NM_021636AB_049405 BC038795 AB083616 AL356953. PREDICTED: Homo sapiens misc_RNA(LOC100128771), miscRNA: XR 037860.1. PREDICTED: Homo sapienshypothetical protein LOC100129705 (LOC100129705), mRNA: XM_001723520.1.PREDICTED: Homo sapiens similar to Fanconi anemia complementation groupD2 protein (LOC100129929), mRNA: XM_001732829. 1Table Headers:SYNONYMS are other recognized names for the gene or transcript.

GenBank and RefSEQ IDs for each of the gems from Tables 1 and 2 appearbelow:

Homo sapiens absent in melanoma 1-Like (AIM1L), mRNA: FLJ1004Q, DKFZp434L1713; FLJ3802G. GenBank/RefSeq IDs: AK000902 NM_001039775 AL451 139CD628848 AK095339 CH471059 BC136870 DR004727 DA439029 AL137264, Homosapiens aminolevulinate, delta-, synthase 2 (ALAS2), nuclear geneencoding mitochondrial protein, transcript variant 3, niRNA: AY532084BP284609 BM677888 AK291589 Λ532085 AY532086 AYS32087 AY532088 AY532089NM_001037967 CR609′889 NM_001037968 NM_000032 CR615092 AY532102 AY532101Z83821 AY532100 CH471154 AK313118 AY532109 AY532108 AY532107 AY532081AY532106 AY532080 AY532105 AY532083 AY532104 AY532082 AY532103 AY532097AY532098 AK290565 AY532095 AY532096 CR593743 X56352 AY532099 AY190322CR613185 AY532094 AY532093 CR626361 AY532092 AY532G91 X60364 CR591435AA778169 AY532069 CR606333 AL020991 CR620984 BP233279 CR606670 AY532079AY532Q78 CR608649 AY532077 AF068624 AY532076 AY532075 AY532074 AY532073BC030230 AY532Q71 AY532072 AY532070 BQ182291 AF130113 Homo sapiensangiopoietin-like 6 (ANGPTL6), mRNA; CR591845 CH471106 AY358276 AB054064BC1 42632 NM_03 1917 CR621970 CR617920 AC020931 AF230330. Homo sapiensbacterieidal/{circumflex over ( )}eriBeability-increasing protein (BPI),mRNA: CH471077 J04739BP306215 CQ800679 Y14219 Y14217 BC03223G Y14218AL583962 Y14215 Y 14216 AK296568 AL359555 AF322588 Y 14220 AK3 15328AI436120 Y14221 Y 14222 Y14223 AL499625 Y14228 Y14229 Y14224 Y14225NM_i⁾0!725 Yl 4226 Y14227 AL39I692 DQ414688 BC040955. Homo sapienschemokine binding protein 2 (CCBP2), mRNA: BC020558 U94888 BT006800CH471055 AK3 13561 DQ229109 AC099329 DA853I59 AY262687 BC008816 BC018716EU832737 AF481959 BC0U588 BC101629 Y12815 BC 112045 BE901 130 NM_001296CR610139 BC01 1631. 1-iomo sapiens CD40 molecule, TNF receptorsuperfamily member 5 (CD40), transcript variant 2, mRNA: CH471077 NMJ52854 CS097717 ΛK222896 BC012419 AB209660 DQ87 1604 AY225405 BM76 1221CR605787 AL035662 EF064754 CS103051 DQ891804 BC064518 CR6 19622 CS103053NM_001250 CS097719 CS097320 AY504960 AJ3G0189 DQ894988 BT0 19901 X60592CS095651 CS097318 CR608994 CS095653. Homo sapiens creatine kinase,mitocliondrial 1B (CKMT1B), nuclear gene encoding mitochondrial protein,mRNA: BC121 002 BC121001 DQ893385 BC108652 AK294908 AK293685 AC011330CR618017 AK293790 AK293961 NM_001015001 AK316319 AK3 16124 DQ896705BC001926 CR605038 AK294735 BU674851 AK094322 AK223365 BM923431J04469AK296271 AK294063 AK296472 NM_020990 CR599868 AK295776 AK3 16052CR616544 CR597545 BT006628 BI1 18196 AK293939 AK308770 CR59951 1BC006467. Homo sapiens cathepsin G (CTSG), mRNA: DQ893067 CR456807BC014460 NM_001911 CR541704 AK225914 DQ896320 AL136018 AI272833J04990CH471078. Homo sapiens defensm, alpha IB (DBFA1B), mRNA: AX405718L12690 NM_004084 AF238378 AF200455 BC069423 X52053 AF233439 M26602BC093791 DQ896798 DQ890546 DQ890545 NM_00ï042500 BC1 12188 M21130. Homosapiens EPS8-like 2 (EPS8L2), mRNA. XM_943956 XM_943960 XM_943963XM_943966: BC101481 BC080636 AK025824 AK094539 AK122903 AK294041BC093878 AK2229G3 AK225311 AK122984 AK025588 NM_022772 BC002474 BC143242AF318331 AP006621 AY074929 CH471158 AK027765. Homo sapiens family withsequence similarity 19 (chemokine (C-C motifj-like), member A2(FAM19A2), mRNA: AL834160 AC078872 BC028403 CR749367 AC078789 CH471054AC020647 AW161831 NM_1 78539 AY3251 15 AC137053 BC050347 BC040286AK123580 AC1304 14. Homo sapiens family with sequence similarity 46,member C (FAM46C), mRNA: BC036516 AL046016 NM_017709 AK091327 CH471 122BC131726 AL365331 AK000209 CD300120. PREDICTED: Homo sapienshypothetical protein FLJ23865 (FLJ23865), mRNA: AK074445. Homo sapiens Gantigen 12F (GAGE12F), mRNA: NC000023.10. Homo sapiensgamma-gluiamyitramferase light chain 1 (GGTLC1), transcript variant A,mRNA: NM_178311 NM_178312 CH471133 BC040904 I, 20491 L20492 AL133466.Homo sapiens glycerol kinase 5 (putative) (GK5), mRNA: DQ894800 BX648681BC032470 BX648359 CH471052 NMJ301039547 AC108679 AK090901 BU628781CR598125 AK310761 AK313792 DQ891607 AK130469 DR000158 AK127641. RST17329Athersys RAGE Library Homo sapiens cDNA, mRNA sequence: Hs.134230. Homosapiens cDNA FLJ1 1554 fis, clone HEMBA1003037: AK021616. yy43f04.slScares melanocyte 2NbHM Homo sapiens cDNA clone IMAGE: 27401 5 3, mRNAsequence: BC000393. Homo sapiens primary neuroblastoma cDNA, clone:Nblal0527, full insert sequence: AB074162. pleckstrin homology domaininteracting protein: AJ303 102 AL049321 CH471051 BX537762 AF3 10250BI548935 AL450327 BCl 37488 BC021905 ΛK 127816 BC008909 AK000712 ALl61957 NM_0 17934 CR600369 BC036479 BC064611 BC081569 DQ924532 AK057039DA5Q0781 AK075124 AL356776 BC144670. oo20c08.xlSoares_NSF_F8_9W_OT_PA_P_S1 Homo sapiens cDNA clone IMAGE: 1566734 3,niRNA sequence: HS.134230. Homo sapiens KIAA1045 (KIAA1045), mRNA: NM_015297. Homo sapiens kelch domain containing 1 (KLHDCl), mRNA: BCl 43597AK127202 AK098735 BC143596 AL591767 NM_172193 BC101595 BC031270 BC101597AF1 11806 AL833437 CH471078. Homo sapiens lipocalin 2 (LCN2), mRNA:AK316217 N79823 CR542092 C!-l47!090 BP272828 X99133 EU644752 X83006S75256 BX644845 BC033089 NM_005564 CA454137 AL590708 AW778875 BM977724AK301694 BF354583. Homo sapiens leueine-ricb repeat-containing Gprotein-coupled receptor 6 (LGR6), transcript variant 3, mRNA: NM_001017404 BC047905 NM_001017403 AK123055 AK027377 BM699180 AF088074CH471067 AY3581 19 AF190501 CQ84081 1 NM_021636 AB04940S BC038795AB083616 AL356953. PREDICTED: Homo sapiens misc_RNA (LOC100128771),miscRNA: XR_037860.1. PREDICTED: Homo sapiens hypothetical proteinLOG100129705 (LOG 100 129705), mRNA: XMJ)01723520.1, PREDICTED: Homosapiens similar to Fanconi anemia complementation group D2 protein(LOC100129929), mRNA: XM_001732829.1 PREDICTED: Homo sapiens misc_RNA{LGC1OO133075), miscRNA: XR_039086.1. PREDICTED: Homo sapiens similar tohCG2024106, transcript variant I (LOCI00134648), mRNA: XM_001724649.1.PREDICTED: Homo sapiens misc_RNA (LOC100134669), miscRNA: XR_038059.1.PREDICTED: Homo sapiens hypothetical protein LOC339047, transcriptvariant 74 (LOC339047), mRNA: XM_001723375 NM_178541 AK124516 BC039707AC137803 BC046145 BC008178 AC138969 AF229069 BC010188 AK126798 AC136619.PREDICTED: Homo sapiens similar to hCGl 809904 (LOC391761), mRNA:XM_373073.2 PREDICTED: Homo sapiens similar to UDP-Gal: betaGlcNAc beta1,3- galactosyltransferase, polypeptide 4 (LOC402377), mRNA: AL599456XR_079072 BP872983 BP872636 AL161911. PREDICTED: Homo sapienshypothetical gene supported by AY0071 55 (LOC439949), mRNA: XM_001723825XM_001 128367 AY007155 XM_0O1129241 AL158043. PREDICTED: Homo sapienssimilar to MHC class II antigen (LOC642073), mRNA: XR_018080.2.PREDICTED: Homo sapiens similar to hCGl 730248 (LQC643037), mRNA:XM_926406 AADB02014322 AP000943 XM_936787. PREDICTED: Homo sapiensregion containing hypothetical protein LOC283970; similar to nuclearpore complex interacting protein, transcript variant 2 (LOC643943),mRNA: XM_934575.1. PREDICTED: Homo sapiens hypothetical proteinLOC645743 (LOC645743), mRNA: XM_928753.1. PREDICTED: Homo sapienssimilar to hCG1774990 (LOC646021), mRNA: XM_001718652.1 PREDICTED: Homosapiens hypothetical LOC646498 (LOC646498), inRNA: NM_001080528 AC135506PREDICTED: Homo sapiens similar to embigin homolog (LOC647121), rnRNA:AL592494 AK128714 NR_003955 NM_198449 AK300860 AK304226 AC035145CR625471 U52054 BC059398 CR621536 AC091833 CH471123 DC309453. PREDICTED:Homo sapiens similar to Neuronal acetylcholine receptor protein, beta-4subunit precursor (LOC649686), mRNA: XM_938759.1. PREDICTED: Homosapiens similar to Iduronate 2-suifatase precursor (Alpha-L- iduronatesulfate sulfatase) (Idursulfase) (LOC653157), mRNA: XM_926258.1.PREDIC′TED: Homo sapiens similar to Neutrophil defensin 1 precursor(HNP-1.) (HP-1) (HP1) (Defensin, alpha 1) (LOC653600), mRNA:XM_928349,1. PREDICTED: Homo sapiens misc_RNA (LOC728522), miscRNA:XR._015716.2. PREDICTED: Homo sapiens similar to ring finger protein 18(LOC732387), mRNA: XM_94 1859.1 Homo sapiens lactotransferrin (LTF),mRNA: S52659 M83205 AY137470 M83202 AF332168 AK303889 CH471055 AF508798U07643 AK316330 M73700 AK292813 DQ522304 U95626 DQ896102 BC015823NMJ)0234 3 X52941 M18642 BC015822 DQ892855 AY493417 AY156717 AK303995AY178998 BC022347 AY165046 EU779935 AK298035 AK093852 AY360320 M93150X53961 AK290859 CD722125 AY875691 AC098613 Homo sapiensmethyltransferase like 7B (METTL7B), mRNA: BC020509 NMJ52637 AY358508AC009779 CH471054 CR595874 AK2901 12 Homo sapiens microRNA 940 (MIR94Q),microRNA: NRJ)30636. 1: NR_030636.1. Homo sapiens myeloperoxidase (MPO),nuclear gene encoding mitochondria! protein, mRNA: S56200 A08802 Z37728M19507 J02694M17170 X15377 M17173 M17174 M17171 M17172 M19508 D14466M17175 M17176 AC004687 BC130476 χ04876 DQ088846 CH471 109 χ64647NM_000250. Homo sapiens myelin transcription factor 1-like (MYTIL),mRNA: BC031690 AL133024 BC042833 AB029029 AB073885 AC1 06046 BC071612BF530661 AC008276 AC009232 BM7 15930 AF036943 BF439754 BC043230 AC093390AC01 1301 AK299571 BC 137273 BCl 50281 BCl 37272 AK307797 AC009471NM_015025 BC1091 13. PREDICTED: Homo sapiens neuroblastoma breakpointfamily, member 1, transcript variant 16 (NBPFl), mRNA: NM_001101663BC094705 AK055895 AL049742 AF379606 AK095030 AF379607 BC034418 CR599564XM_—002346226 CR608846 BC169317 BC169318 BC169316 BC094841 DB300232AF380582 NM_001037675 BC086308 AL1 17237 AF3 80580 NM_183372 BC063799BX546486 BC027348 AL592284 NM_001 039703 AC026900 AK302413 AF379624NM_015383 AF379626 AF379627 AF379628 AK294944 XM_001726946 AK092351AF379620 AF379621 AF3 79622 AF3 79623 AK054850 AL359I76 XM_001717398AF379615 AF379616 AF379613 AF131738 AF379614 AL355149 AF379619 AE1 38796BX5 1 1041 AK290302 AF379617 AL050141 AF379618 BC021 111 AF379611AF379612 AY894574 BC010124 AY894573 BCl 48331 AY894572 AL040349 AY894571AY894570 BC071995 AY894579 AY894578 AY894577 AI,592307 AY894576 AY894575AL137798 AK290142 AI865471 AF419617 XM_0017I. 5810 AF419616 AF419619AF419618 AK095459 AF379632 AY894583 AF3 79631 A(.356004 AY894582AF379634 AY894585 BCl 10431 AF379630 AY894581 AK125792 AY894580 AL139152BCl 67783 AK294414 AF379635 NM_017940 AF420437 BQ890458 AK00Q726BC136292 CR600619 AL954711 BC071723 AF161426 B{umlaut over (l)}552657AB051480 CR610345 AK097180 BC023087 BX648497 AL022240 AL832622 AB033071AY894561 BC013805 AY894563 AY894562 BC066930 AY894565 AY894567 AY894566BX538005 AY894569 AY894568 BX842679 NM_173638 DQ786323 AK299360NM_001170755 BC093404 AK{umlaut over (l)}23260, Homo sapiensneuroblastoma breakpoint family, member 7 (NBPF7), mRNA: NM_001047980.1.Homo sapiens S′-nucleotidase domain containing 2 (NT5DC2), mRNA:DA013090 DB265466 AK022504 CH471055 AW510639 BC047747 DR003312 AC1 12215CR612874 BC014550 BM982658 NM_022908 AK092469 NM_001 134231 AF131781AK023995. Homo sapiens neurotrophin 3 (NTP3), mRNA: AC137627 BC107075X53-S3S CH471 116 AC007848 CN267386 AK293895 AW190653 CD672441 BC069773BU939830 NM_002527 M37763 NM_001 102654 CR541906 M61180. Homo sapiensphospholipase C-Iike 1 (PLCL1), mRNA: D42108 AC092599 NM_(3011 14661CH471063 ACI 09589 DA414853 AC013478 AC020719 BC101531 BP340710 BX537442NM_006226 BC1 1 1985 DA193701 AGO11997 T87219 AK127514 AK302673. Homosapiens processing of precursor 5, ribonuclease P/MRP subunit (S.cerevisiae) (POP5), transcript variant 2, mRNA: AJ306296 CB995722AF070660 CH471054 AA918619 AC063943 BU155588 AK223206 BI668578 BC012505NM_015918 AK303144 B{umlaut over (l)}549018 NM_J98202 CR625634 API17232. Homo sapiens PR domain containing 12 (PRDM12), mRNA: NM_021619CH471090 BC172255 AY004252 AL359092. Homo sapiens resistin (RETN), mRNA:NM_020415 BC069302 AC008763 BC101560 DQ301958 AF205952 AY207314 BC101554AF352730 AF290874 AY359066 CH471139 AF32308L Homo sapiens regulator ofG-protein signaling 20 (RGS20), transcript variant 1, mRNA: AF493940AC100821 AC113194 AF060877 BC015614 AK094503 NM_G03702 AY046538 CR622266BC063490 CR602987 CH471068 AF074979 NM_170587 BC031328 CR594448 BCG18618AF366057 AF366056 AF366055 AP366054. Homo sapiens ribosomal proteinL10-iike (RPL10L), mRNA: AK130863 NM_080746 AL591768 AB063608 BC0663I2AM392934 AB063605 CH471078 BC014310. Homo sapiens selenoprotein P,plasma, 1 (SEPP1), transcript variant 1, mRNA: BC05I919 Z11793 AK094640NM_005410 AC008945 AK225801 BC040075 NM_001093726 NM_o01085486 BC005244AL833145 B1463468 AK096125 BC046152 AK311392 ALU365 19 BC015875 CR607609CH471119 CR607105 DO022288 BC030009. Homo sapiens splicing factor 1(SFl). transcript variant 4, mRNA: AP001462 AK301803 BC034451 BQ230035CH471076 AK296237 AJ000OS2 EU832634 AJ000051 BC000773 L49345 BC069273D26120 EU832710 BX095574 D26121 D26122 AK293753 BC008080 AK299705 BCOl1657 BC008724 BC038446 BC032676 BC020217 NM_201995 NM_201997 Y08765MM_201998 L49380 Y08766 CR600558 NM_004630 BU624935 CR615439. Homosapiens SH3-domain GRB2-like (endopbilin) interacting protein 1 (SGiPl),mRNA: AK125044 BC040516 AL139147 NM_032291 AL354978 AL356913 DA801276CH471059 BX640813 AB210039 CR622 14 1 AK298970 EU831934 ALi36561AL391820 AK090697 AK29994I AM392887 CR749541 EU832029. Homo sapiens SHC(Src homology 2 domain containing) transforming protein 3 (SHC3), mRNA:D84361 BC026314 AL160054 DQ896663 AL353150 NM_016848 BX641I39, Homosapiens solute carrier family 22 (organic cation transporter), member 2(SLC22A2), mRNA: NM_003058.2 NMJ)03058 BC039899 CH471051 AL162582AB075951 X98333 CR618035 AK290787 AJ251885 BC030978, Homo sapiens solutecarrier family 7 (cationic amino acid transporter, y + system), member 5(SLC7A5), mRNA: CH471114 ABO18542 NM_003486 CR625513 CR601601 AM182888BC039692 BC042600 CR594130 AB017908 DQ896766 AF077866 DQ893338 ABO18009AB023721 BC014177 AC126696 AF104032 M80244 BC1 14608. Homo sapienssolute carrier family 7 (cationic amino acid transporter, y + system),member 6 (SLC7A6), transcript variant 2, mRNA: CR623370 BC028216AC020978 NM_001076785 DQ894203 CR601248 CR613948 DQ891023 CR749475 AK310866 AK311610 D87432 BP242589 BC1 13100 NM_003983 CH471092 CR606394AK310602 CR592629 CR749291. Homo sapiens small nucleolar RNA, C/D box 13(SNORD13), small micleoiai- RNA: NR_003041 AC091144, Homo sapienssynaptophysin-like 1 (SYPL1), transcript variant 1, mRNA: BG719851CH471070 NMJ82715 AK292230 CB989012 BQ429369 X68194 BC020938 X61382EU831812 NM_006754 S72481 AA969654 BC061887 BC016835 AC005095 CR616230AK128279 CH236947 EU831889. Homo sapiens tripartite motif-containing 34(TRIM34), transcript variant 2, mRNA: AK293295 NM_130390 BC1 56770DA847894 BM989990 AK027664 AF220144 BG721109 AF220143 AC015691NM_001003827 BG776117 BC140722 AK298301 NM_021616 CX760618 CR624250AK023210 CR749260 AK3 16289 BC065575 AK290172 BC136871 NM_ 130389AL583914 CH471064 BC047564 NM_001003818 NM_QQ1003819 NM_058166 AK316178AB039903 AB039902 AK027876 AF220030 AB039904, Homo sapiens V-set andtransmembrane domain containing 2A (VSTM2A), mRNA: NM_182546.2. Homosapiens zinc finger protein 461 (ZNF461), mRNA: NM_153257 AB209279AB021641 BC028631 AY329493 BX649031 AK292834 DQ894367 AK299370 AC074138DQ891 185. Homo sapiens zinc finger protein 485 (7NF485), mRNA: CQ783574BC014161 NMJ45312 AK074679 AL645634 CH471 160 BX648149 AK3 13328AK299707. PREDICTED: Homo sapiens misc_RNA (ZNF788), miscRNA:XRJ)41527.1, Homo sapiens zinc finger protein 830 (ZNF830), mRNA:NM_052857.3. PREDICTED: Homo sapiens zinc finger protein 90 (HTF9)(ZNF90), mRNA: CR614976 NM_007138 CN386341 BC137211 CR593334 AC006539CH471106 AK298173 AC011447 BX1 02781 M61870. Homo sapiens alkalinephosphatase, liver/bone/kidney (ALPL), transcript variant 1, mRNA:AL592309 ABO11406 BC066116 ABO12643 BC136325 NM_0G0478 NM_001 127501AL359815 X53750 BC021289 AB209814 D87880 D87882 D87881 AK298085 M24429BC126165 M24428 BC110909 D87877 D87887 D87876 CH471134 D87888 D87879D8N89 D87878 D87883 AK3 12667 D87884 DA625627 D87875 D87885 D87874D87886 DA631560 M24435 M24434 M24433 M24432 BC090861 M24431 M24430AK293184 M24439 M24438 M24437 M24436 AK295608 X14174 AK097413. Homosapiens interleukin 8 receptor, beta (IL8RB). mRNA; U1 1869 DA670033U11866 AK290906 DQ895671 NM_001168298 DA674925 i.19593 AB032733 AC124768AB032734 U11873 U11872 DQ893661.

Example 2: Determination of Selected Biomarkers by Reverse Transcriptase(RT) and Droplet Digital Polymerase Chain Reaction (ddPCR)

In the prior example, 5 patients were categorized as lower respiratoryinfections (LRI), but analysis of the ease reports indicates that thepatients with the lowest biomarker levels, especially DEFA1, wereactually unlikely to have had a pulmonary infection, One patient clearlyhad a pulmonary embolism (PE) secondary to air travel, and anothersubject was actually a healthcare worker with a mild upper respiratory(UR) condition that could have been allergic.

In order to determine more fully whether the biomarkers had value indetecting pulmonary infections, 4 additional subjects were obtained, andthey were compared with 3 subjects with known hernias, surgicallyconfirmed, and 5 subjects presenting for coronary catheterization but noknown pulmonary infection or distress. The 2 patients from Example 1with PE and UR. condition were excluded, because they were not, in fact,suspected LRI/pneumonia, yielding 7 suspected LRI, and 8 controlsubjects,

Methods: The Tempus blood RNA preservative tubes were frozen at −80 C asin prior studies, and then thawed and total nucleic acid was isolated bythe manufacturer's protocol. The total nucleic acids were treated withDNAse, as above, and the remaining RNA was concentrated and quantifiedas above. A specific amount of RNA (500 ng) was taken into a reversetranscriptase (RT) reaction using BioRad iScript (RNAse H+) with randomhexamers primers, according to the manufacturer's procedure. From the RTreaction, the resulting cDNA was diluted 1:15 with, water to make astock cDNA solution from each patient for further analysis. The stockcDNA from each patient was diluted 1:10 or 1:20 into the PGR reactionand <mixed with reagents for ddPCR. as specified by the manufacturer. InddPCR, the RNA strands in aqueous solution are emulsified in oil diluentto create ‘droplets’ of extremely small volumes of aqueous solution suchthat, on average, only a single strand of any particular RNA will becontained within each droplet. Each droplet also contains polymerase,nucleotides, and the specific primers that are employed b quantify aspecific RNA target (eg DEFA1) and a fluorochrome that detects the PCRamplimer (EvaGreen), thus making each droplet a small reaction vesselfor PGR amplification. Any droplet that contain a DEI⁷A1 RNA transcriptwill produce a positive fluorescent droplet, while droplets that arenegative for DEFA1 will produce little or no fluorescence. Thus, thenumber of positive droplets will be directly proportional to theabsolute quantity of the RNA target in the sample and can be calculatedusing Bayesian statistics and a Poisson distribution.

The ddPCR analysis was conducted on the 7 suspected LRI, and 8 controlsubjects, using 3 biomarkers (DEFA1, ALPL, IL8RB) and one invariantcontrol (ACTB). The ALPL and IL8RB markers are included here because thelungs can also be affected by biofilm infections, although that couldnot be determined in the present 7 suspected LRI subjects. Thus, DEFA1is the exemplary biomarker that should be sensitive to a pulmonaryinfection. The absolute quantity of the transcripts was quantified byddPCR and then corrected for any dilutions that were employed (10× or20×), and then the levels of the biomarkers were expressed as a % ofACTB invariant control level for each sample.

Results: A s shown in FIG. 1 , the level of the DEFA1 biomarker ismarkedly higher in suspected LRI/pneumo patients (n=7) versus non-LRIcontrols (n−8) (LRI=81.6%, (sem=42.4), vs Con=8.6¾(3.Q¾), t-testp<0.05). Likewise, the ALPL biomarker was also higher in LRI thancontrols, (LRI=2.7%(0.6%) vs Con=0.7%(0.09%), p<0.01). Neither IL8RB(L-R1===6.0%(1.5%) vs Con=<S.6%(1.0%), p>0.35), nor the combination ofALPL+IL8RB (LR1-8.7%(1.4%) v s Con=7.2%(1.1%), p>0.20) was significantlydifferent between groups. Two of the subjects with suspected pneumoniashowed relatively normal DEFA1 levels and so their charts were reviewed,In one case, it was discovered that the patient had been on antibioticsfor 7 days prior to admission and blood sampling, and in the other case,the patient had pneumonitis due to amiodarone toxicity, with a cough,fever, and apparent infiltrates on X-ray, but showed a normal whiteblood cell count and had negative bacterial cultures, suggesting thatthe patient may not have had an active pulmonary infection at the timeof sampling, although the clinical suspicion was reasonable.

Example 3: Other Embodiments of a Neutrophil Activation Test forRespiratory Infections

These further embodiments utilize the RNA and protein biomarkersdisclosed in Examples 1 and 2 above, and disclose alternative methods bywhich they could be quantified in clinically relevant instances forhumans and other living species.

Identification of RNA Biomarkers in Immune Cells without the Need forLysis and RNA Purification.

There is a huge, worldwide, unmet medical need for a simple, rapid,inexpensive test that reports whether the patient's immune system isresponding to a pathogen. In the U.S., some of this diagnostic need isfulfilled with advanced imaging tests, such as X-rays, CT, and MRI, butin developing countries, and rural areas of developed nations,diagnostic tools are limited to a thermometer, stethoscope, and possiblya white cell count.

A cell-based assay. In one embodiment of the present invention, termed‘cell-based assay’, the said biomarkers would be quantitated on ceils,or cell fractions derived from the biological sample. The biologicalsample would include any samples previously described, such as blood,sputum, lavage, spinal fluid, etc, but the method of biomarkerdetermination would not require the lysis of the cells containedtherein. Rather, the cells within the sample would be captured by any ofa variety of means known to persons skilled in the art, and then the RNAor protein levels of said biomarkers would be quantitated on the cellsdirectly. In this method, the cells can be captured with, or withoutcentrifugation, which has significant advantages because centrifugationis not always available in non-hospital settings. Further, the number ofceils required would be quite small, for instance in the range of 10,000cells, which can be obtained from just microliters of blood.Venipuncture is painful and distressing, especially to children, and soa finger stick would be belter tolerated, but still provide an adequatesample of cells for analysis.

The overall design of the test is to conduct the cell-based assay onsmall volumes of blood, such as obtained from a venipuncture or fingerstick. In one embodiment, the blood is drawn via capillary action into aflat chamber, which is separated into several smaller chambers. Wedisclose herein an exemplary method using just 2 cells types,neutrophils and T cells. Other types of cells can also be used. In oneembodiment of the invention, each flow chamber is coated with specificantibodies to surface markers for neutrophils (CD 16b) or T cells (CD4)so that the desired cell is trapped by adhesion in that well [3; 4].After an incubation period of about 10-20 minutes, the unbound cells areflushed away with buffer contained in the fluidic pack,

Biomarker detection. As described, any of a variety of conventionalmethods are used to immobilize cell-specific antibodies on a glasscoverslip or other suitable surface to capture cells from whole blood orother relevant, biological samples. After washing away unbound cells,the bound cells are contacted with any of the analytical tools describedfor measuring the specific biomarkers, such as, for example, resistins(RSTN), defensins (DEFA1), and myeloperoxidases (MPQ). These analyticaldetection methods share the common trait that they bind to the targetbiomarker, and produce a detectable signal when bound to the biomarker.

Engineering: Any handheld automated microfluidic device and a smartphonebased imager can be adapted for use in a method as described herein [5;6], Suitable commercially available devices can also be used.

Bioinfonnatie: Once the handheld imager captures the detectable signalof said biomarkers in the cells, where for example the signal is DEFA1mRNA level, it is then simple integration to compute the signalintensity per cell and ratio the level to the invariant control (e.g.,ACTB) Because multiple channels can be used, or multiple signals on thesame channel, it is clear that-multiple biomarkers can be quantitated onthe same biological sample. In such a case, the biological markers couldbe combined to create a metric of neutrophil activation, such as,(RSTN+DEFA1+MPO)/ACTB=neutrophil activation level.

Alternatively or in addition, a ligase-based assay can be used toquantitate the RNA levels in suitable samples. Likewise, it istechnically feasible to conduct RNA or cDNA sequencing reactions withincells, as described in the scientific literature.

Instead of, or in addition to, assays using RNA markers, either thelevel of protein or protein enzymatic activity can be used as thebiomarker. For example, the cell-based assay described above can beadapted to detect RSTN or DEFA1 protein level, by a fluorescentantibody, or an enzymatic activity such MPO activity. Because separatechambers can be used for each type of reaction, one embodiment employs ahybrid test in which, for example, RSTN RNA is detected in one chamber,DEFA1 antigen in another, and MPO activity in a third. This can beaccomplished without the need for obtaining additional clinical samples.

REFERENCES FOR BACKGROUND AND EXAMPLES

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From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make changes andmodificadoiis of the invention to adapt it to various usage andconditions and to utilize the present invention to its fullest extent.The preceding embodiments are to be construed as merely illustrative,and not limiting of the scope of the invention in any way whatsoever.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompositions and methods of use thereof described herein. Suchequivalents are considered b be within the scope of this invention andare covered by the following claims. Those skilled in the art will alsorecognize that all combinations of embodiments described herein arewithin the scope of the invention.

What is claimed is:
 1. A method comprising administering a treatment fora lower respiratory infection to a subject, wherein a nucleic acidsample from the subject has been assayed to detect the expression levelof at least the biomarkers of alkaline phosphatase liver isoform (ALPL),interleukin-8 receptor beta (IL8RB), and defensin-alpha 1, and thesubject was determined to have an at least three-fold increase ordecrease in the expression level of the biomarkers as compared to atleast one invariant control marker, wherein the at least three-foldincrease or decrease in the expression level of the biomarkers isindicative of a lower respiratory infection, wherein the treatment forthe lower respiratory infection comprises antibiotics, antivirals,anti-inflammatories, or any combination thereof.
 2. The method of claim1, wherein the at least three-fold increase or decrease in theexpression level of the biomarkers as compared to the at least oneinvariant control marker has been determined by a process comprising:obtaining a biological sample from said subject, wherein the biologicalsample comprises nucleic acids; detecting the expression level of saidbiomarkers alkaline phosphatase liver isoform (ALPL), interleukin-8receptor beta (IL8RB), and defensin-alpha 1 in the biological sample;and determining said increase or decrease in the expression level of thebiomarkers by comparing the expression level of said biomarkers to saidat least one invariant control marker.
 3. The method of claim 2, furthercomprising calculating a biomarker infection score from said increase ordecrease in the expression level of said biomarkers and comparing saidbiomarker infection score to a control score.
 4. The method of claim 2,wherein said determining an increase or decrease in the expression levelof said biomarkers comprises using a multivariate prediction model todetermine if a pattern of expression of said biomarkers is indicative ofa lower respiratory infection.
 5. The method of claim 2, wherein saidbiological sample is a blood sample.
 6. The method of claim 5, whereinsaid obtaining a biological sample further comprises isolating immunecells from said blood sample.
 7. The method of claim 6, wherein saidimmune cells are neutrophils, T-cells, or a combination thereof.
 8. Themethod of claim 2, wherein detecting the expression level of saidbiomarkers comprises measuring RNA levels of said biomarkers.
 9. Themethod of claim 8, wherein measuring RNA levels comprises usingfluorescently-labeled probes complementary to said biomarkers, aligase-based assay, reverse transcriptase and polymerase chain reaction,RNA sequencing, or cDNA microarray.
 10. The method of claim 1, whereinsaid at least one invariant control marker is selected from the groupconsisting of Spi-B transcription factor, protein phosphatase 1regulatory subunit 21 (PPP1R21, KLRAQ1), mitogen-activated proteinkinase kinase kinase 7 (MAP3K7, TAK1), olfactory receptor family 51subfamily member 1 (OR51M1), BCL2 antagonist/killer (BAK1), adenosinedeaminase, RNA-specific (ADAR1), and actin-beta (ACTB).